Processes for preparing tubulysins

ABSTRACT

Processes for preparing tubulysins and derivatives thereof are described. In addition, processes for preparing unnatural tubulysins are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit, under 35 U.S.C. §119(e), ofU.S. Provisional Application No. 61/771,429, filed Mar. 1, 2013, andU.S. Provisional Application 61/793,082, filed Mar. 15, 2013, theentirety of each of the disclosures of which are hereby incorporatedherein by reference.

TECHNICAL FIELD

The invention described herein pertains to processes for preparingtubulysins and derivatives thereof. In particular, the processes pertainto the preparation of unnatural tubulysins.

BACKGROUND AND SUMMARY OF THE INVENTION

The tubulysins are members of a new class of natural products isolatedfrom myxobacterial species (F. Sasse, et al., J. Antibiot. 2000, 53,879-885). As cytoskeleton interacting agents, the tubulysins are mitoticpoisons that inhibit tubulin polymerization and lead to cell cyclearrest and apoptosis (H. Steinmetz, et al., Chem. Int. Ed. 2004, 43,4888-4892; M. Khalil, et al., ChemBioChem. 2006, 7, 678-683; G. Kaur, etal., Biochem. J. 2006, 396, 235-242). Tubulysins are extremely potentcytotoxic molecules, exceeding the cell growth inhibition of anyclinically relevant traditional chemotherapeutic e.g. epothilones,paclitaxel, and vinblastine. Furthermore, they are potent againstmultidrug resistant cell lines (A. Domling, et al., Mol. Diversity.2005, 9, 141-147). These compounds show high cytotoxicity tested againsta panel of cancer cell lines with IC₅₀ values in the low picomolarrange; thus, they are of interest as potential anticancer therapeutics.Accordingly, processes for preparing tubulysins, including non-naturallyoccurring tubulysins are needed.

Tubulysins are described herein. Structurally, tubulysins often includelinear tetrapeptoid backbones, including illustrative compounds havingformula T or AT

and pharmaceutically acceptable salts thereof; wherein

Ar₁ is optionally substituted aryl;

R₁ is hydrogen, alkyl, arylalkyl or a pro-drug forming group;

R₂ is selected from the group consisting of optionally substituted alkyland optionally substituted cycloalkyl;

R₁₂ is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which isoptionally substituted;

R₄ is optionally substituted alkyl or optionally substituted cycloalkyl;

R₃ is optionally substituted alkyl;

R₅ and R₆ are each independently selected from the group consisting ofoptionally substituted alkyl and optionally substituted cycloalkyl;

R₇ is optionally substituted alkyl; and

n is 1, 2, 3, or 4.

Another illustrative group of tubulysins described herein are moreparticularly comprised of one or more non-naturally occurring orhydrophobic amino acid segments, such as N-methyl pipecolic acid (Mep),isoleucine (Ile),

and analogs and derivatives of each of the foregoing. A common featurein the molecular architecture of the more potent natural occurringtubulysins is the acid and/or base sensitive N-acyloxymethyl substituent(or a N,O-acetal of formaldehyde) represented by R2-C(O) in the formula(T).

Another illustrative group of tubulysins described herein are thosehaving formula 1.

Structures of Several Natural Tubulysins

Tubulysin R_(A) R₂ A OH CH₂CH(CH₃)₂ B OH CH₂CH₂CH₃ C OH CH₂CH₃ D HCH₂CH(CH₃)₂ E H CH₂CH₂CH₃ F H CH₂CH₃ G OH CH═C(CH₃)₂ H H CH₃ I OH CH₃

A total synthesis of tubulysin D possessing C-terminal tubuphenylalanine(R_(A)=H) (H. Peltier, et al., J. Am. Chem. Soc. 2006, 128, 16018-16019)has been reported. Recently, a modified synthetic protocol toward thesynthesis of tubulysin B (R_(A)=OH) (O. Pando, et at., Org. Lett. 2009,11, 5567-5569) has been reported. However, attempts to follow thepublished procedures to provide larger quantities of tubulysins wereunsuccessful, being hampered in part by low yields, difficult to removeimpurities, the need for expensive chromatographic steps, and/or thelack of reproducibility of several steps. The interest in usingtubulysins for anticancer therapeutics accents the need for reliable andefficient processes for preparing tubulysins, and analogs andderivatives thereof. Described herein are improved processes for makingtubulysins, or analogs or derivatives thereof, including compounds offormula (AT).

In one illustrative embodiment of the invention, processes for preparingtubulysins, or analogs or derivatives thereof, including compounds offormula (AT). The processes include one or more steps described herein.In another embodiment, a process is described for preparing a compoundof formula B, wherein R₅ and R₆ are as described in the variousembodiments herein, such as each being independently selected fromoptionally substituted alkyl or optionally substituted cycloalkyl; andR₈ is C1-C6 n-alkyl; wherein the process comprises the step of treatinga compound of formula A with a silylating agent, such as triethylsilylchloride, and a base, such as imidazole in an aprotic solvent.

It is to be understood that R₅ and R₆ may each include conventionalprotection groups on the optional substituents.

In another embodiment, a process is described for preparing a compoundof formula C, wherein R₅ and R₆ are as described in the variousembodiments herein, such as each being independently selected fromoptionally substituted alkyl or optionally substituted cycloalkyl; R₈ isC1-C6 n-alkyl; and R₂ is as described in the various embodiments herein,such as being selected from optionally substituted alkyl or optionallysubstituted cycloalkyl; wherein the process comprises the step oftreating a compound of formula B with a base and a compound of theformula ClCH₂OC(O)R₂ in an aprotic solvent at a temperature belowambient temperature, such as in the range from about −78° C. to about 0°C.; wherein the molar ratio of the compound of the formula ClCH₂OC(O)R₂to the compound of formula B from about 1 to about 1.5.

It is to be understood that R₂, R₅ and R₆ may each include conventionalprotection groups on the optional substituents.

In another embodiment, a process is described for preparing a compoundof formula D, wherein R₅ and R₆ are as described in the variousembodiments herein, such as being selected from optionally substitutedalkyl or optionally substituted cycloalkyl; R₈ is C1-C6 n-alkyl; R₂ isas described in the various embodiments herein, such as being selectedfrom optionally substituted alkyl or optionally substituted cycloalkyl;and R₇ is optionally substituted alkyl; wherein the process comprisesthe steps of

a) preparing a compound of formula (E1) where X₁ is a leaving group froma compound of formula E; and

b) treating a compound of formula C under reducing conditions in thepresence of the compound of formula E1.

It is to be understood that R₂, R₅, R₆, and R₇ may each includeconventional protection groups on the optional substituents.

In another embodiment, a process is described for preparing a compoundof formula AF, wherein R₅ and R₆ are as described in the variousembodiments herein, such as being selected from optionally substitutedalkyl or optionally substituted cycloalkyl; R₂ is as described in thevarious embodiments herein, such as being selected from optionallysubstituted alkyl or optionally substituted cycloalkyl; and R₇ isoptionally substituted alkyl; wherein the process comprises the step ofcontacting compound D with an alcohol, R₁₂OH, where R₁₂ is alkyl,alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,arylalkyl or heteroarylalkyl, each of which is optionally substituted;and a transesterification catalyst. In another embodiment, thetransesterification catalyst is selected from the group consisting of(R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂, (R₁₃)₂SnS,(R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃ is independently selectedfrom alkyl, arylalkyl, aryl, or cycloalkyl, each of which is optionallysubstituted. In another embodiment, the transesterification catalyst is(R₁₃)₂SnO. Illustrative examples of R₁₃ are methyl, n-butyl. n-octyl,phenyl, o-MeO-phenyl, p-MeO phenyl, phenethyl, and benzyl.

It is to be understood that R₅, R₆, R₁₂, and R₇ may each includeconventional protection groups on the optional substituents.

In another embodiment, a process is described for preparing a compoundof formula AG, wherein R₅ and R₆ are as described in the variousembodiments herein, such as being selected from optionally substitutedalkyl or optionally substituted cycloalkyl; R₂ is as described in thevarious embodiments herein, such as being selected from optionallysubstituted alkyl or optionally substituted cycloalkyl; and R₇ isoptionally substituted alkyl; wherein the process comprises the step ofcontacting compound F with an alcohol, R₁₂OH, where R₁₂ is alkyl,alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,arylalkyl or heteroarylalkyl, each of which is optionally substituted;and a transesterification catalyst. In another embodiment, thetransesterification catalyst is selected from the group consisting of(R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂, (R₁₃)₂SnS,(R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃ is independently selectedfrom alkyl, arylalkyl, aryl, or cycloalkyl, each of which is optionallysubstituted. In another embodiment, the transesterification catalyst is(R₁₃)₂SnO. Illustrative examples of R₁₃ are methyl, n-butyl. n-octyl,phenyl, o-MeO-phenyl, p-MeO phenyl, phenethyl, and benzyl.

It is to be understood that R₂, R₅, R₆, R₇, and R₁₂ may each includeconventional protection groups on the optional substituents.

In another embodiment, a process is described for preparing a compoundof formula BG, wherein R₅ and R₆ are as described in the variousembodiments herein, such as being selected from optionally substitutedalkyl or optionally substituted cycloalkyl; R₂ is as described in thevarious embodiments herein, such as being selected from optionallysubstituted alkyl or optionally substituted cycloalkyl; R₁₂ is asdescribed in the various embodiments herein, such as being selected fromalkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl,aryl, arylalkyl or heteroarylalkyl, each of which is optionallysubstituted; and R₇ is optionally substituted alkyl; wherein the processcomprises the step of contacting compound AF with a metal hydroxide orcarbonate. Illustrative examples of a metal hydroxide or carbonateinclude LiOH, Li₂CO₃, NaOH, Na₂CO₃, KOH, K₂CO₃, Ca(OH)₂, CaCO₃, Mg(OH)₂,MgCO₃, and the like.

It is to be understood that R₅, R₆, R₇, and R₁₂ may each includeconventional protection groups on the optional substituents.

In another embodiment, a process is described for preparing a compoundof formula AH, wherein R₅ and R₆ are as described in the variousembodiments herein, such as being selected from optionally substitutedalkyl or optionally substituted cycloalkyl; R₂ and R₄ are as describedin the various embodiments herein, such as being selected fromoptionally substituted alkyl or optionally substituted cycloalkyl; R₁₂is as described in the various embodiments herein, such as beingselected from alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which isoptionally substituted; and R₇ is optionally substituted alkyl; whereinthe process comprises the step of treating a compound of formula BG withan acylating agent of formula R₄C(O)X₂, where X₂ is a leaving group.

It is to be understood that R₄, R₅, R₆, and R₇ may each includeconventional protection groups on the optional substituents.

In another embodiment, a process is described for preparing a tubulysinof formula (AT), wherein Ar₁ is aryl or heteroaryl each of which isoptionally substituted; R₁ is hydrogen, optionally substituted alkyl,optionally substituted arylalkyl or a pro-drug forming group; R₅ and R₆are as described in the various embodiments herein, such as beingselected from optionally substituted alkyl or optionally substitutedcycloalkyl; R₃ is optionally substituted alkyl; R₂ and R₄ are asdescribed in the various embodiments herein, such as being selected fromoptionally substituted alkyl or optionally substituted cycloalkyl; R₁₂is as described in the various embodiments herein, such as beingselected from alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which isoptionally substituted; and R₇ is optionally substituted alkyl; whereinthe process comprises the step of forming an active ester intermediatefrom a compound of formula AH; and reacting the active esterintermediate with a compound of the formula I to give a compound of theformula AT.

It is to be understood that Ar₁, R₁, R₂, R₄, R₅, R₆, R₇, and R₁₂ mayeach include conventional protection groups on the optionalsubstituents.

In another embodiment, a process is described for preparing a tubulysinlinker derivative of formula (TL-2), wherein Ar₁ is optionallysubstituted aryl or optionally substituted heteroaryl; Ar₂ is optionallysubstituted aryl or optionally substituted heteroaryl; L is selectedfrom the group consisting of

where p is an integer from about 1 to about 3, m is an integer fromabout 1 to about 4, and * indicates the points of attachment;R^(a), R^(b), and R are each independently selected in each instancefrom the group consisting of hydrogen and alkyl; or at least two ofR^(a), R^(b), or R are taken together with the attached carbon atoms toform a carbocyclic ring;

R_(Ar) represents 0 to 4 substituents selected from the group consistingof amino, or derivatives thereof, hydroxy or derivatives thereof, halo,thio or derivatives thereof, alkyl, haloalkyl, heteroalkyl, aryl,arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl,heteroarylheteroalkyl, nitro, sulfonic acids and derivatives thereof,carboxylic acids and derivatives thereof; R₁ is hydrogen, optionallysubstituted alkyl, optionally substituted arylalkyl or a pro-drugforming group; R₅ and R₆ are as described in the various embodimentsherein, such as being selected from optionally substituted alkyl oroptionally substituted cycloalkyl; R₃ is optionally substituted alkyl;R₂ and R₄ are as described in the various embodiments herein, such asbeing selected from optionally substituted alkyl or optionallysubstituted cycloalkyl; and R₇ is optionally substituted alkyl; whereinthe process comprises the step of contacting compound TL, with analcohol, R₁₂OH, where R₁₂ is alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, eachof which is optionally substituted; and a transesterification catalyst.In one embodiment the transesterification catalyst is TFA. In anotherembodiment, the transesterification catalyst is selected from the groupconsisting of (R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂,(R₁₃)₂SnS, (R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃ is independentlyselected from alkyl, arylalkyl, aryl, or cycloalkyl, each of which isoptionally substituted. In another embodiment, the transesterificationcatalyst is (R₁₃)₂SnO. Illustrative examples of R₁₃ are methyl, n-butyl.n-octyl, phenyl, o-MeO-phenyl, p-MeO phenyl, phenethyl, and benzyl. Itis to be understood that Ar₁, Ar₂, R₁, R₂, R₄, R₅, R₆, R₇, and R₁₂ mayeach include conventional protection groups on the optionalsubstituents.

In another embodiment, a process is described for preparing a tubulysinlinker derivative of formula (TL-2), wherein Ar₁ is optionallysubstituted aryl or optionally substituted heteroaryl; Ar_(e) isoptionally substituted aryl or optionally substituted heteroaryl; L isselected from the group consisting of

wherein

p is an integer from about 1 to about 3, m is an integer from about 1 toabout 4, and * indicates the points of attachment;

R^(a), R^(b), and R are each independently selected in each instancefrom the group consisting of hydrogen and alkyl; or at least two ofR^(a), R^(b), or R are taken together with the attached carbon atoms toform a carbocyclic ring;

R_(Ar) represents 0 to 4 substituents selected from the group consistingof amino, or derivatives thereof, hydroxy or derivatives thereof, halo,thio or derivatives thereof, alkyl, haloalkyl, heteroalkyl, aryl,arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl,heteroarylheteroalkyl, nitro, sulfonic acids and derivatives thereof,carboxylic acids and derivatives thereof;

R₁ is hydrogen, optionally substituted alkyl, optionally substitutedarylalkyl or a pro-drug forming group; R₅ and R₆ are as described in thevarious embodiments herein, such as being selected from optionallysubstituted alkyl or optionally substituted cycloalkyl; R₃ is optionallysubstituted alkyl; R₂ and R₄ are as described in the various embodimentsherein, such as being selected from optionally substituted alkyl oroptionally substituted cycloalkyl; and R₇ is optionally substitutedalkyl;

wherein the process comprises the step of forming an active esterintermediate from a compound of formula AH; and reacting the activeester intermediate with a compound of the formula IL to give a compoundof the formula TL-2.

In another embodiment, the process described in any of the embodimentsdescribed herein wherein Ar₁ is optionally substituted aryl isdescribed.

In another embodiment, the process described in any of the embodimentsdescribed herein wherein Ar₁ is optionally substituted heteroaryl isdescribed.

It is to be understood that Ar₁, Ar₂, R₁, R₁₂, R₃, R₄, R₅, R₆, and R₇may each include conventional protection groups on the optionalsubstituents in any of the embodiments described herein.

DETAILED DESCRIPTION

In one embodiment, a process is described for preparing a compound offormula B, wherein R₅ and R₆ are as described in the various embodimentsherein, such as being selected from optionally substituted alkyl oroptionally substituted cycloalkyl; and R₈ is C1-C6 n-alkyl; wherein theprocess comprises the step of treating a compound of formula A withtriethylsilyl chloride and imidazole in an aprotic solvent.

In the previously reported preparations of the intermediate silyl etherof formula 2, use of a large excess of triethylsilyltrifluoromethylsulfonate (TESOTf) and lutidine is described (see, forexample, Peltier, et al., 2006). It was found that the reported processmakes it necessary to submit the product of the reaction to achromatographic purification step. Contrary to that reported, it hasbeen surprisingly discovered herein that the less reactive reagent TESClmay be used. It has also been surprisingly discovered herein thatalthough TESCl is a less reactive reagent, it may nonetheless be used innearly stoichiometric amounts in the processes described herein. It isappreciated herein that the use of the less reactive TESCl may also beadvantageous when the process is performed on larger scales, wherehigher reactivity reagents may represent a safety issue. It has alsobeen discovered that the use of TESCl in nearly stoichiometric amountsrenders the chromatographic purification step unnecessary. In analternative of the embodiment, the process is performed withoutsubsequent purification. In another alternative of the foregoingembodiments, and each additional embodiment described herein, R₅ isisopropyl. In another alternative of the foregoing embodiments, and eachadditional embodiment described herein, R₆ is sec-butyl. In anotheralternative of the foregoing embodiments, and each additional embodimentdescribed herein, R₈ is methyl. In another alternative of the foregoingembodiments, and each additional embodiment described herein, the silylether is TES.

In an illustrative example of the processes described herein, a processfor preparing the silyl ether 2 in high yield is described whereincompound 1 is treated with 1.05 equivalent of TESCl and 1.1 equivalentof imidazole.

In one alternative of the foregoing example, the compound 2 is notpurified by chromatography.

In another embodiment, a process is described for preparing a compoundof formula C, wherein R₅ and R₆ are each independently selected from thegroup consisting of optionally substituted alkyl and optionallysubstituted cycloalkyl; R₈ is C1-C6 n-alkyl; and R₂ is selected from thegroup consisting of optionally substituted alkyl and optionallysubstituted cycloalkyl; wherein the process comprises the step oftreating a compound of formula B with from about 1 equivalent to about1.5 equivalent of base and from about 1 equivalent to about 1.5equivalent of a compound of the formula ClCH₂OC(O)R₂ in an aproticsolvent at a temperature from about −78° C. to about 0° C.

In another embodiment, the process of the preceding embodiment isdescribed wherein the compounds of formulae B and C have thestereochemistry shown in the following scheme for B′ and C′.

In another illustrative embodiment, the process of any one of thepreceding embodiments is described wherein about 1 equivalent to about1.3 equivalent of a compound of the formula ClCH₂OC(O)R₂ is used. Inanother illustrative example, the process of any one of the precedingembodiments is described, wherein about 1.2 equivalent of a compound ofthe formula ClCH₂OC(O)R₂ is used. In another illustrative example, theprocess of any one of the preceding embodiments is described wherein R₂is n-propyl. In another alternative of the foregoing embodiments, andeach additional embodiment described herein, R₂ is CH₂CH(CH₃)₂,CH₂CH₂CH₃, CH₂CH₃, CH═C(CH₃)₂, or CH₃.

In an illustrative example of the processes described herein, a processfor preparing the N,O-acetal 3 is described. In another illustrativeexample, compound 2 is treated with 1.1 equivalent of potassiumhexamethyldisilazane (KHMDS) and 1.2 equivalent of chloromethylbutanoate in a nonprotic solvent at about −45° C. In anotherillustrative example, the product formed by any of the precedingexamples may be used without chromatographic purification.

In another embodiment, a process is described for preparing a compoundof formula D, wherein R₅ and R₆ are each independently selected from thegroup consisting of optionally substituted alkyl and cycloalkyl; R₈ isC1-C6 n-alkyl; R₂ is selected from the group consisting of optionallysubstituted alkyl and cycloalkyl; and R₇ is optionally substitutedalkyl; wherein the process comprises the steps of

a) preparing a compound of formula (E1) where X₁ is a leaving group froma compound of formula E; and

b) treating a compound of formula C under reducing conditions with thecompound of formula E1.

In one illustrative example, a mixture of compound 3 and thepentafluorophenyl ester of D-N-methyl-pipecolic acid is reduced using H₂and a palladium-on-charcoal catalyst (Pd/C) to yield compound 4. It hasbeen discovered herein that epimerization of the active ester ofpipecolic acid can occur during reaction or during its preparation orduring the reduction under the previously reported reaction conditions.For example, contrary to prior reports indicating that epimerizationdoes not occur (see, for example, Peltier, 2006), upon repeating thosereported processes on a larger scale it was found here that substantialamounts of epimerized compounds were formed. In addition, it wasdiscovered herein that substantial amounts of rearrangement productsformed by the rearrangement of the butyryl group to compound 8 wereformed using the reported processes. Finally, it was discovered hereinthat the typical yields of the desired products using the previouslyreported processes were only about half of that reported. It has beendiscovered herein that using diisopropylcarbodiimide (DIC) and shortreaction times lessens that amount of both the unwanted by-productresulting from the epimerization reaction and the by-product resultingfrom the rearrangement reaction. In another alternative of the foregoingembodiments, and each additional embodiment described herein, n is 3. Inanother alternative of the foregoing embodiments, and each additionalembodiment described herein, R₇ is methyl.

In one illustrative example, it was found that limiting the reactiontime for the preparation of pentafluorophenyl D-N-methyl-pipecolate toabout 1 hour lessened the formation of the diastereomeric tripeptide 9.It has also been discovered that using dry 10% Pd/C as catalyst, ratherthan a more typically used wet or moist catalyst, lessens the amount ofepimer 9 formed during the reduction. It has also been discovered thatusing dry 10% P/C and/or shorter reaction times also lessens theformation of rearranged amide 8.

It has been previously reported that removal of the protecting groupfrom the secondary hydroxyl group leads to an inseparable mixture of thedesired product 5 and a cyclic O,N-acetal side-product as shown in thefollowing scheme.

Further, upon repeating the reported process, it has been discoveredherein that removal of the methyl ester using basic conditions, followedby acetylation of the hydroxyl group leads to an additional previouslyunreported side-product, iso-7. That additional side-product isdifficult to detect and difficult to separate from the desired compound7. Without being bound by theory, it is believed herein that iso-7results from rearrangement of the butyrate group from theN-hydroxymethyl group to the secondary hydroxyl group, as shown below.

In another embodiment, a process is described for preparing a compoundof formula AF, wherein R₅ and R₆ are as described in the variousembodiments herein, such as being selected from optionally substitutedalkyl or optionally substituted cycloalkyl; R₂ is as described in thevarious embodiments herein, such as being selected from optionallysubstituted alkyl or optionally substituted cycloalkyl; and R₇ isoptionally substituted alkyl; wherein the process comprises the step ofcontacting compound D with an alcohol, R₁₂OH, where R₁₂ is alkyl,alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,arylalkyl or heteroarylalkyl, each of which is optionally substituted;and a transesterification catalyst. In another embodiment thetransesterification catalyst is trifluoroacetic acid (TFA). In anotherembodiment, the transesterification catalyst is selected from the groupconsisting of (R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂,(R₁₃)₂SnS, (R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃ is independentlyselected from alkyl, arylalkyl, aryl, or cycloalkyl, each of which isoptionally substituted. In another embodiment, the transesterificationcatalyst is (R₁₃)₂SnO. Illustrative examples of R₁₃ are methyl, n-butyl.n-octyl, phenyl, o-MeO-phenyl, p-MeO phenyl, phenethyl, and benzyl.

It is to be understood that R₅, R₆, R₁₂, and R₇ may each includeconventional protection groups on the optional substituents.

In an illustrative example, compound 4 is heated with an alcohol anddi-n-butyltin oxide at about 100° C. to yield ether 10. It isappreciated that a co-solvent may be present. In one embodiment, themolar ratio (tin oxide)/(compound 10) is about 0.01 to about 0.30, orabout 0.02 to about 0.20, or about 0.05 to about 0.15, or about 0.05 toabout 0.10

In another embodiment, a process is described for preparing a compoundof formula BG, wherein R₅ and R₆ are as described in the variousembodiments herein, such as being selected from optionally substitutedalkyl or optionally substituted cycloalkyl; R₂ is as described in thevarious embodiments herein, such as being selected from optionallysubstituted alkyl or optionally substituted cycloalkyl; R₁₂ is asdescribed in the various embodiments herein, such as being selected fromalkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl,aryl, arylalkyl or heteroarylalkyl, each of which is optionallysubstituted; and R₇ is optionally substituted alkyl; wherein the processcomprises the step of contacting compound AF with a metal hydroxide orcarbonate. Illustrative examples of a metal hydroxide or carbonateinclude LiOH, Li₂CO₃, NaOH, Na₂CO₃, KOH, K₂CO₃, Ca(OH)₂, CaCO₃, Mg(OH)₂,MgCO₃, and the like.

It is to be understood that R₅, R₆, R₇, and R₁₂ may each includeconventional protection groups on the optional substituents.

In an illustrative example, compound 10 is treated with LiOH.H₂O in amixture of THF and water at about room temperature to yield compound 11.It is appreciated that the THF may be replaced with other solvents.

In another embodiment, a process is described for preparing a compoundof formula AH, wherein R₅ and R₆ are each independently selected fromthe group consisting of optionally substituted alkyl and optionallysubstituted cycloalkyl; R₂ and R₄ are independently selected from thegroup consisting of optionally substituted alkyl and optionallysubstituted cycloalkyl; and R₇ is optionally substituted alkyl; whereinthe process comprises the step of treating a compound of formula BG withan acylating agent of formula R₄C(O)X₂, where X₂ is a leaving group. Itis appreciated that the resulting product may contain varying amounts ofthe mixed anhydride of compound AH and R₄CO₂H. In another embodiment,the process described in the preceding embodiment further comprises thestep of treating the reaction product with water to prepare AH, free ofor substantially free of anhydride. In another embodiment, the processof the preceding embodiments wherein X₂ is R₄CO₂, is described. Inanother embodiment, the process of any one of the preceding embodimentswherein R₄ is C1-C4 alkyl is described. In another alternative of theforegoing embodiments, and each additional embodiment described herein,R₄ is methyl. In another embodiment, the process of any one of thepreceding embodiments wherein R₆ is sec-butyl is described. In anotherembodiment, the process of any one of the preceding embodiments whereinR₇ is methyl is described. In another embodiment, the process of any oneof the preceding embodiments wherein R₅ is iso-propyl is described.

In an illustrative example, compound 11 is treated with acetic anhydridein pyridine. It is appreciated that the resulting product may containvarying amounts of the mixed anhydride of 12 and acetic acid. In anotherembodiment, treatment of the reaction product resulting from thepreceding step with water in dioxane yields compound 12, free of orsubstantially free of anhydride. It is to be understood that othersolvents can be substituted for dioxane in the hydrolysis of theintermediate mixed anhydride. Alternatively, the step may be performedwithout solvent.

In another embodiment, a process is described for preparing a tubulysinof formula (AT), wherein Ar₁ is optionally substituted aryl; R₁ ishydrogen, optionally substituted alkyl, optionally substituted arylalkylor a pro-drug forming group; R₅ and R₆ are as described in the variousembodiments herein, such as being selected from optionally substitutedalkyl or optionally substituted cycloalkyl; R₃ is optionally substitutedalkyl; R₂ and R₄ are as described in the various embodiments herein,such as being selected from optionally substituted alkyl or optionallysubstituted cycloalkyl; R₁₂ is as described in the various embodimentsherein, such as being selected from alkyl, alkenyl, alkynyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl orheteroarylalkyl, each of which is optionally substituted; and R₇ isoptionally substituted alkyl; wherein the process comprises the step offorming an active ester intermediate from a compound of formula AH; andreacting the active ester intermediate with a compound of the formula Ito give a compound of the formula AT.

It is to be understood that Ar₁, R₁, R₂, R₄, R₅, R₆, R₇, and R₁₂ mayeach include conventional protection groups on the optionalsubstituents.

In one embodiment, compound AH is treated with an excess amount ofactive ester forming agent and pentafluorophenol to form thepentafluorophenol ester of compound AH, followed by removal of theexcess active ester forming agent prior to the addition of compound I.In another alternative of the foregoing embodiments, and each additionalembodiment described herein, Ar₁ is phenyl. In another alternative ofthe foregoing embodiments, and each additional embodiment describedherein, Ar₁ is substituted phenyl. In another alternative of theforegoing embodiments, and each additional embodiment described herein,Ar₁ is 4-substituted phenyl. In another alternative of the foregoingembodiments, and each additional embodiment described herein, Ar₁ isR_(A)-substituted phenyl. In another alternative of the foregoingembodiments, and each additional embodiment described herein, Ar₁ is4-hydroxyphenyl, or a hydroxyl protected form thereof. In anotheralternative of the foregoing embodiments, and each additional embodimentdescribed herein, R₃ is methyl. In another alternative of the foregoingembodiments, and each additional embodiment described herein, R₁ ishydrogen.

In an illustrative example, compound 12 is treated with an excess amountof a polymeric version of a carbodiimide and pentafluorophenol to formthe pentafluorophenyl ester of 12, the polymeric carbodiimide is removedby filtration; and amino acid (S)-tubutyrosine is added to the solutionto yield the tubulysin, compound 13. In another embodiment, the processof any one of the preceding embodiments wherein the polymericcarbodiimide is polystyrene-CH₂—N═C═N-cyclohexane (PS-DCC) is described.

In another embodiment, a compound AF is described wherein R₁₂, R₅, R₆,and R₇ are as described in the any of the embodiments described herein.

In another embodiment, the following compound is described wherein R₁₂,R₅, R₆, R₇ and R₈ are as described in the any of the embodimentsdescribed herein.

In another embodiment, the compound having formula 10 is described.

In another embodiment a compound BG, is described, wherein R₁₂, R₅, R₆,and R₇ are as described in any of the embodiments described herein.

In another embodiment, compound 11 is described.

In another embodiment, compound 7 is described.

In another embodiment, a compound AH is described wherein R₄ is Me andR₁₂, R₅, R₆, and R₇ are as described in any of the embodiments describedherein; and the compound H is free of or substantially free of thecompound H wherein R₄ and R₂ are both Me.

In another alternative of the foregoing embodiments, and each additionalembodiment described herein, R₅ is isopropyl.

In another alternative of the foregoing embodiments, and each additionalembodiment described herein, R₆ is sec-butyl.

In another alternative of the foregoing embodiments, and each additionalembodiment described herein, R₈ is methyl.

In another alternative of the foregoing embodiments, and each additionalembodiment described herein, R₂ is CH₂CH(CH₃)₂, CH₂CH₂CH₃, CH₂CH₃,CH═C(CH₃)₂, or CH₃.

In another alternative of the foregoing embodiments, and each additionalembodiment described herein, R₁₂ is CH₂CH═CH₂, or CH₂(CH₂)nCH₃, where nis 1, 2, 3, 4, 5, or 6.

In another alternative of the foregoing embodiments, and each additionalembodiment described herein, R₁₂ is CH₂CH═CH₂, CH₂CH₂CH₂CH₃, orCH₂CH₂CH₂CH₂CH₃.

In another alternative of the foregoing embodiments, and each additionalembodiment described herein, n is 3.

In another alternative of the foregoing embodiments, and each additionalembodiment described herein, R₇ is methyl.

In another alternative of the foregoing embodiments, and each additionalembodiment described herein, R₄ is methyl.

In another alternative of the foregoing embodiments, and each additionalembodiment described herein, Ar₁ is phenyl. In another alternative ofthe foregoing embodiments, and each additional embodiment describedherein, Ar₁ is substituted phenyl. In another alternative of theforegoing embodiments, and each additional embodiment described herein,Ar₁ is 4-substituted phenyl. In another alternative of the foregoingembodiments, and each additional embodiment described herein, Ar₁ isR_(A)-substituted phenyl. In another alternative of the foregoingembodiments, and each additional embodiment described herein, Ar₁ is4-hydroxyphenyl, or a hydroxyl protected form thereof.

In another alternative of the foregoing embodiments, and each additionalembodiment described herein, R₃ is methyl.

In another alternative of the foregoing embodiments, and each additionalembodiment described herein, R₁ is hydrogen.

Illustrative embodiments of the invention are further described by thefollowing enumerated clauses:

1. A process for preparing a compound of the formula

or a pharmaceutically acceptable salt thereof; wherein Ar₁ is optionallysubstituted aryl or optionally substituted heteroaryl;

R₁ is hydrogen, alkyl, arylalkyl or a pro-drug forming group;

R₂ is selected from the group consisting of optionally substituted alkyland optionally substituted cycloalkyl;

R₁₂ is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which isoptionally substituted;

R₃ is optionally substituted alkyl;

R₄ is optionally substituted alkyl or optionally substituted cycloalkyl;

R₅ and R₆ are each independently selected from the group consisting ofoptionally substituted alkyl and optionally substituted cycloalkyl; R₇is optionally substituted alkyl; and n is 1, 2, 3, or 4;

wherein the process comprises the step of treating a compound of formulaA with triethylsilyl chloride and imidazole in an aprotic solvent, whereR₈ is C1-C6 unbranched alkyl

or

the step of treating a compound of formula B with a base and a compoundof the formula ClCH₂OC(O)R₂ in an aprotic solvent at a temperature fromabout −78° C. to about 0° C.; wherein the molar ratio of the compound ofthe formula ClCH₂OC(O)R₂ to the compound of formula B from about 1 toabout 1.5, where R₈ is C1-C6 unbranched alkyl

or

the steps of a) preparing a compound of formula (E1), where X₁ is aleaving group, from a compound of formula E

andb) treating a compound of formula C under reducing conditions in thepresence of the compound of formula E1, where R₈ is C1-C6 unbranchedalkyl

or

the step of contacting compound D with an alcohol, R₁₂OH, where R₁₂ isalkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl,aryl, arylalkyl or heteroarylalkyl, each of which is optionallysubstituted; and a transesterification catalyst selected from TFA or thegroup consisting of (R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO,(R₁₃)₂SnCl₂, (R₁₃)₂SnS, (R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃ isindependently selected from alkyl, arylalkyl, aryl, or cycloalkyl, eachof which is optionally substituted;

or

the step of treating the compound AF with a metal hydroxide or a metalcarbonate;

or

the step of treating a compound of formula BG with an acylating agent offormula R₄C(O)X₂, where X₂ is a leaving group

or

the steps of c) forming an active ester intermediate from a compound offormula AH

andd) reacting the active ester intermediate with a compound of the formulaI

or

one or more combinations thereof.

2. The process of clause 1 wherein Ar₁ is optionally substituted aryl.

3. The process of clause 1 wherein Ar₁ is optionally substitutedheteroaryl.

4. A process for preparing a compound having formula (TL-2)

wherein

L is selected from the group consisting of

where p is an integer from about 1 to about 3, m is an integer fromabout 1 to about 4, and * indicates the points of attachment;

R^(a), R^(b), and R are each independently selected in each instancefrom the group consisting of hydrogen and alkyl; or at least two ofR^(a), R^(b), or R are taken together with the attached carbon atoms toform a carbocyclic ring;

R_(Ar) represents 0 to 4 substituents selected from the group consistingof amino, or derivatives thereof, hydroxy or derivatives thereof, halo,thio or derivatives thereof, alkyl, haloalkyl, heteroalkyl, aryl,arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl,heteroarylheteroalkyl, nitro, sulfonic acids and derivatives thereof,carboxylic acids and derivatives thereof;

wherein the process comprises the step of contacting a compound havingformula (TL)

with R₁₂OH, where R₁₂ is alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, eachof which is optionally substituted; and a transesterification catalystselected from TFA or the group consisting of (R₁₃)₈Sn₄O₂(NCS)₄,(R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂, (R₁₃)₂SnS, (R₁₃)₃SnOH, and(R₁₃)₃SnOSn(R₁₃)₃, where R₁₃ is independently selected from alkyl,arylalkyl, aryl, or cycloalkyl, each of which is optionally substituted.

5. The process of any one of the preceding clauses wherein R₄ isoptionally substituted alkyl.

6. The process of any one of the preceding clauses comprising the stepof treating a compound of formula A with triethylsilyl chloride andimidazole in an aprotic solvent, where R₈ is C1-C6 unbranched alkyl

7. The process of any one of the preceding clauses comprising the stepof treating a compound of formula B with a base and a compound of theformula ClCH₂OC(O)R₂ in an aprotic solvent at a temperature from about−78° C. to about 0° C.; wherein the molar ratio of the compound of theformula ClCH₂OC(O)R₂ to the compound of formula B from about 1 to about1.5, where R₈ is C1-C6 unbranched alkyl

8. The process of any one of the preceding clauses comprising the stepsof

a) preparing a compound of formula (E1), where X₁ is a leaving group,from a compound of formula E

andb) treating a compound of formula C under reducing conditions in thepresence of the compound of formula E1, where R₈ is C1-C6 unbranchedalkyl

9. The process of any one of the preceding clauses comprising the stepof treating compound D with an alcohol, R₁₂OH, where R₁₂ is alkyl,alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,arylalkyl or heteroarylalkyl, each of which is optionally substituted;and a transesterification catalyst selected from TFA or the groupconsisting of (R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂,(R₁₃)₂SnS, (R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃ is independentlyselected from alkyl, arylalkyl, aryl, or cycloalkyl, each of which isoptionally substituted;

10. The process of any one of the preceding clauses comprising the stepof treating the compound AF with a metal hydroxide or a metal carbonate;

11. The process of any one of the preceding clauses comprising the stepof treating a compound of formula BG with an acylating agent of formulaR₄C(O)X₂, where X₂ is a leaving group

12. The process of any one of the preceding clauses comprising the stepsof

c) forming an active ester intermediate from a compound of formula AH

andd) reacting the active ester intermediate with a compound of the formulaI

13. A process for preparing a compound of the following formula

the process comprising the step of contacting a compound of the formula

with an acid and R₁₂OH, wherein R₂, R₅, R₆, R₈, and R₁₂ are as describedin any of the embodiments described herein.

14. A process for preparing a compound of the following formula

the process comprising the step of contacting a compound of the formula

with a transesterification catalyst selected from the group consistingof (R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂, (R₁₃)₂SnS,(R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃ and R₁₂OH, wherein R₂, R₅,R₆, R₈, R₁₂, and R₁₃ are as described in any of the embodimentsdescribed herein.

15. A process for preparing a compound of the following formula

the process comprising the step of contacting a compound of the formula

with a base and R₁₂OCH₂X, where X is Cl or Br; and wherein R₂, R₅, R₆,R₈, R₁₂, and R₁₃ are as described in any of the embodiments describedherein. In another embodiment, R₁₂OCH₂X is n-C₅H₁₁OCH₂Br.

16. A process for preparing a compound of the following formula

the process comprising the step of contacting a compound of the formula

with an acid and R₁₂OH, wherein R₁₄ is Et₃Si or R₄C(O), and R₂, R₄, R₅,R₆, R₈, and R₁₂ are as described in any of the embodiments describedherein.

17. A process for preparing a compound of the following formula

the process comprising the step of contacting a compound of the formula

with a transesterification catalyst selected from the group consistingof (R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂, (R₁₃)₂SnS,(R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃; and R₁₂OH, wherein R₂, R₅,R₆, R₈, R₁₂, and R₁₃ are as described in any of the embodimentsdescribed herein.

18. A process for preparing a compound of the following formula

the process comprising the step of contacting a compound of the formula

with an acid and R₁₂OH, wherein n, R₂, R₃, R₄, R₅, R₆, R₇, R₈, Ar₁, Ar₂,L and R₁₂ are as described in any of the embodiments described herein.

19. The process of any one of the preceding clauses wherein R₁ ishydrogen, benzyl, or C1-C4 alkyl.

19A. The process of any one of the preceding clauses wherein R₁ ishydrogen.

20. The process of any one of the preceding clauses wherein R₂ is C1-C8alkyl or C3-C8 cycloalkyl.

20A. The process of any one of the preceding clauses wherein R₂ isn-butyl.

20B. The process of any one of the preceding clauses wherein R₂ isCH₂CH(CH₃)₂, CH₂CH₂CH₃, CH₂CH₃, CH═C(CH₃)₂, or CH₃.

21. The process of any one of the preceding clauses wherein R₃ is C1-C4alkyl.

21A. The process of any one of the preceding clauses wherein R₃ ismethyl.

22. The process of any one of the preceding clauses wherein Ar₁ isphenyl or hydroxyphenyl.

22A. The process of any one of the preceding clauses wherein Ar₁ is4-hydroxyphenyl.

23. The process of any one of the preceding clauses wherein R₄ is C1-C8alkyl or C3-C8 cycloalkyl.

23A. The process of any one of the preceding clauses wherein R₄ ismethyl.

24. The process of any one of the preceding clauses wherein R₅ isbranched C3-C6 or C3-C8 cycloalkyl.

24A. The process of any one of the preceding clauses wherein R₅ isiso-propyl.

25B. The process of any one of the preceding clauses wherein R₅ issec-butyl.

26. The process of any one of the preceding clauses wherein R₆ isbranched C3-C6 or C3-C8 cycloalkyl.

27. The process of any one of the preceding clauses wherein R₇ is C1-C6alkyl.

27A. The process of any one of the preceding clauses wherein R₇ ismethyl.

28. The process of any one of the preceding clauses wherein R₁₂ isCH₂CH═CH₂, or CH₂(CH₂)nCH₃, where n=1, 2, 3, 4, 5, or 6.

28A. The process of any one of the preceding clauses wherein R₁₂ isCH₂CH═CH₂, CH₂CH₂CH₂CH₃, or CH₂CH₂CH₂CH₂CH₃.

29. The process of any one of the preceding clauses wherein Ar₁ issubstituted phenyl.

29A. The process of any one of the preceding clauses wherein Ar₁ is4-substituted phenyl.

29B. The process of any one of the preceding clauses wherein Ar₁ isR_(A)-substituted phenyl.

29C. The process of any one of the preceding clauses wherein Ar₁ is4-hydroxyphenyl, or a hydroxyl protected form thereof.

30. The process of any one of the preceding clauses wherein R₁₃ isCH₂CH₂CH₂CH₃.

31. The process of any one of the preceding clauses wherein the metalhydroxide is LiOH.

32. A compound of the formula

or a pharmaceutically acceptable salt thereof; wherein Ar₁ is optionallysubstituted aryl;

R₁ is hydrogen, alkyl, arylalkyl or a pro-drug forming group;

R₂ is selected from the group consisting of optionally substituted alkyland optionally substituted cycloalkyl;

R₁₂ is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which isoptionally substituted;

R₃ is optionally substituted alkyl;

R₄ is optionally substituted alkyl or optionally substituted cycloalkyl;

R₅ and R₆ are each independently selected from the group consisting ofoptionally substituted alkyl and optionally substituted cycloalkyl; R₇is optionally substituted alkyl; and n is 1, 2, 3, or 4.

33. A compound of formula

35. A compound of formula

35. A compound of formula

36. A compound of formula

37. The compound of any one of the preceding clauses wherein R₁ ishydrogen, benzyl, or C1-C4 alkyl.

37A. The compound of any one of the preceding clauses wherein R₁ ishydrogen.

38. The compound of any one of the preceding clauses wherein R₂ is C1-C8alkyl or C3-C8 cycloalkyl.

38A. The compound of any one of the preceding clauses wherein R₂ isn-butyl.

38B. The compound of any one of the preceding clauses wherein R₂ isCH₂CH(CH₃)₂, CH₂CH₂CH₃, CH₂CH₃, CH═C(CH₃)₂, or CH₃.

39. The compound of any one of the preceding clauses wherein R₃ is C1-C4alkyl.

39A. The compound of any one of the preceding clauses wherein R₃ ismethyl.

40. The compound of any one of the preceding clauses wherein Ar₁ isphenyl or hydroxyphenyl.

40A. The compound of any one of the preceding clauses wherein Ar₁ is4-hydroxyphenyl.

40B. The compound of any one of the preceding clauses wherein Ar₁ issubstituted phenyl.

40C. The compound of any one of the preceding clauses wherein Ar₁ is4-substituted phenyl.

40D. The compound of any one of the preceding clauses wherein Ar₁ isR_(A)-substituted phenyl.

40E. The compound of any one of the preceding clauses wherein Ar₁ is4-hydroxyphenyl, or a hydroxyl protected form thereof.

41. The compound of any one of the preceding clauses wherein R₄ is C1-C8alkyl or C3-C8 cycloalkyl.

41A. The compound of any one of the preceding clauses wherein R₄ ismethyl.

42. The compound of any one of the preceding clauses wherein R₅ isbranched C3-C6 or C3-C8 cycloalkyl.

42A. The compound of any one of the preceding clauses wherein R₅ isiso-propyl.

42B. The compound of any one of the preceding clauses wherein R₅ issec-butyl.

43. The compound of any one of the preceding clauses wherein R₆ isbranched C3-C6 or C3-C8 cycloalkyl.

44. The compound of any one of the preceding clauses wherein R₇ is C1-C6alkyl.

44A. The compound of any one of the preceding clauses wherein R₇ ismethyl.

45. The compound of any one of the preceding clauses wherein R₁₂ isCH₂CH═CH₂, or CH₂(CH₂)nCH₃, where n=1, 2, 3, 4, 5, or 6.

45A. The compound of any one of the preceding clauses wherein R₁₂ isCH₂CH═CH₂, CH₂CH₂CH₂CH₃, or CH₂CH₂CH₂CH₂CH₃.

46. The compound selected from the group consisting of

where n=1, 2, 3, 4, 5, or 6.

In any of the embodiments described herein, the acid selected for theconversion of the NCH₂OC(O)R₂ moiety to the NCH₂OR₁₂ moiety is TFA.

In any of the embodiments described herein, the catalyst selected forthe conversion of the NCH₂OC(O)R₂ moiety to the NCH₂OR₁₂ moiety is(n-Bu)₂SnO.

It is to be understood that as used herein, the term tubulysin refersboth collectively and individually to the naturally occurringtubulysins, and the analogs and derivatives of tubulysins. Illustrativeexamples of a tubulysin are shown in Table 1.

As used herein, the term tubulysin generally refers to the compoundsdescribed herein and analogs and derivatives thereof. It is also to beunderstood that in each of the foregoing, any correspondingpharmaceutically acceptable salt is also included in the illustrativeembodiments described herein.

It is to be understood that such derivatives may include prodrugs of thecompounds described herein, compounds described herein that include oneor more protection or protecting groups, including compounds that areused in the preparation of other compounds described herein.

In addition, as used herein the term tubulysin also refers to prodrugderivatives of the compounds described herein, and including prodrugs ofthe various analogs and derivatives thereof. In addition, as usedherein, the term tubulysin refers to both the amorphous as well as anyand all morphological forms of each of the compounds described herein.In addition, as used herein, the term tubulysin refers to any and allhydrates, or other solvates, of the compounds described herein.

It is to be understood that each of the foregoing embodiments may becombined in chemically relevant ways to generate subsets of theembodiments described herein. Accordingly, it is to be furtherunderstood that all such subsets are also illustrative embodiments ofthe invention described herein.

The compounds described herein may contain one or more chiral centers,or may otherwise be capable of existing as multiple stereoisomers. It isto be understood that in one embodiment, the invention described hereinis not limited to any particular stereochemical requirement, and thatthe compounds, and compositions, methods, uses, and medicaments thatinclude them may be optically pure, or may be any of a variety ofstereoisomeric mixtures, including racemic and other mixtures ofenantiomers, other mixtures of diastereomers, and the like. It is alsoto be understood that such mixtures of stereoisomers may include asingle stereochemical configuration at one or more chiral centers, whileincluding mixtures of stereochemical configuration at one or more otherchiral centers.

Similarly, the compounds described herein may include geometric centers,such as cis, trans, (E)-, and (Z)-double bonds. It is to be understoodthat in another embodiment, the invention described herein is notlimited to any particular geometric isomer requirement, and that thecompounds, and compositions, methods, uses, and medicaments that includethem may be pure, or may be any of a variety of geometric isomermixtures. It is also to be understood that such mixtures of geometricisomers may include a single configuration at one or more double bonds,while including mixtures of geometry at one or more other double bonds.

As used herein, the term aprotic solvent refers to a solvent which doesnot yield a proton to the solute(s) under reaction conditions.Illustrative examples of nonprotic solvents are tetrahydrofuran (THF),2,5-dimethyl-tetrahydrofuran, 2-methyl-tetrahydrofuran, tetrahydropyran,diethyl ether, t-butyl methyl ether, dimethyl formamide,N-methylpyrrolidinone (NMP), and the like. It is appreciated thatmixtures of these solvents may also be used in the processes describedherein.

As used herein, an equivalent amount of a reagent refers to thetheoretical amount of the reagent necessary to transform a startingmaterial into a desired product, i.e. if 1 mole of reagent istheoretically required to transform 1 mole of the starting material into1 mole of product, then 1 equivalent of the reagent represents 1 mole ofthe reagent; if X moles of reagent are theoretically required to convert1 mole of the starting material into 1 mole of product, then 1equivalent of reagent represents X moles of reagent.

As used herein, the term active ester forming agent generally refers toany reagent or combinations of reagents that may be used to convert acarboxylic acid into an active ester.

As used herein, the term active ester generally refers to a carboxylicacid ester compound wherein the divalent oxygen portion of the ester isa leaving group resulting in an ester that is activated for reactingwith compounds containing functional groups, such as amines, alcohols orsulfhydryl groups. Illustrative examples of active ester-formingcompounds are N-hydroxysuccinimide, N-hydroxyphthalimide, phenolssubstituted with electron withdrawing groups, such as but not limited to4-nitrophenol, pentafluorophenol, N,N′-disubstituted isoureas,substituted hydroxyheteroaryls, such as but not limited to 2-pyridinols,1-hydroxybenzotriazoles, 1-hydroxy-7-aza-benzotriazoles, cyanomethanol,and the like. Illustratively, the reaction conditions for displacing theactive ester with a compound having an amino, hydroxy or thiol group aremild. Illustratively, the reaction conditions for displacing the activeester with a compound having an amino, hydroxy or thiol group areperformed at ambient or below ambient temperatures. Illustratively, thereaction conditions for displacing the active ester with a compoundhaving an amino, hydroxy or thiol group are performed without theaddition of a strong base. Illustratively, the reaction conditions fordisplacing the active ester with a compound having an amino, hydroxy orthiol group are performed with the addition of a tertiary amine base,such as a tertiary amine base having a conjugate acid pKa of about 11 orless, about 10.5 or less, and the like.

As used herein, the term “alkyl” includes a chain of carbon atoms, whichis optionally branched. As used herein, the term “alkenyl” and “alkynyl”includes a chain of carbon atoms, which is optionally branched, andincludes at least one double bond or triple bond, respectively. It is tobe understood that alkynyl may also include one or more double bonds. Itis to be further understood that in certain embodiments, alkyl isadvantageously of limited length, including C₁-C₂₄, C₁-C₁₂, C₁-C₈,C₁-C₆, and C₁-C₄. Illustratively, such particularly limited length alkylgroups, including C₁-C₈, C₁-C₆, and C₁-C₄ may be referred to as loweralkyl. It is to be further understood that in certain embodimentsalkenyl and/or alkynyl may each be advantageously of limited length,including C₂-C₂₄, C₂-C₁₂, C₂-C₈, C₂-C₆, and C₂-C₄. Illustratively, suchparticularly limited length alkenyl and/or alkynyl groups, includingC₂-C₈, C₂-C₆, and C₂-C₄ may be referred to as lower alkenyl and/oralkynyl. It is appreciated herein that shorter alkyl, alkenyl, and/oralkynyl groups may add less lipophilicity to the compound andaccordingly will have different pharmacokinetic behavior. In embodimentsof the invention described herein, it is to be understood, in each case,that the recitation of alkyl refers to alkyl as defined herein, andoptionally lower alkyl. In embodiments of the invention describedherein, it is to be understood, in each case, that the recitation ofalkenyl refers to alkenyl as defined herein, and optionally loweralkenyl. In embodiments of the invention described herein, it is to beunderstood, in each case, that the recitation of alkynyl refers toalkynyl as defined herein, and optionally lower alkynyl. Illustrativealkyl, alkenyl, and alkynyl groups are, but not limited to, methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,pentyl, 2-pentyl, 3-pentyl, neopentyl, hexyl, heptyl, octyl, and thelike, and the corresponding groups containing one or more double and/ortriple bonds, or a combination thereof.

As used herein, the term “cycloalkyl” includes a chain of carbon atoms,which is optionally branched, where at least a portion of the chain incyclic. It is to be understood that cycloalkylalkyl is a subset ofcycloalkyl. It is to be understood that cycloalkyl may be polycyclic.Illustrative cycloalkyl include, but are not limited to, cyclopropyl,cyclopentyl, cyclohexyl, 2-methylcyclopropyl, cyclopentyleth-2-yl,adamantyl, and the like. As used herein, the term “cycloalkenyl”includes a chain of carbon atoms, which is optionally branched, andincludes at least one double bond, where at least a portion of the chainin cyclic. It is to be understood that the one or more double bonds maybe in the cyclic portion of cycloalkenyl and/or the non-cyclic portionof cycloalkenyl. It is to be understood that cycloalkenylalkyl andcycloalkylalkenyl are each subsets of cycloalkenyl. It is to beunderstood that cycloalkyl may be polycyclic. Illustrative cycloalkenylinclude, but are not limited to, cyclopentenyl, cyclohexylethen-2-yl,cycloheptenylpropenyl, and the like. It is to be further understood thatchain forming cycloalkyl and/or cycloalkenyl is advantageously oflimited length, including C₃-C₂₄, C₃-C₁₂, C₃-C₈, C₃-C₆, and C₅-C₆. It isappreciated herein that shorter alkyl and/or alkenyl chains formingcycloalkyl and/or cycloalkenyl, respectively, may add less lipophilicityto the compound and accordingly will have different pharmacokineticbehavior.

As used herein, the term “heteroalkyl” includes a chain of atoms thatincludes both carbon and at least one heteroatom, and is optionallybranched. Illustrative heteroatoms include nitrogen, oxygen, and sulfur.In certain variations, illustrative heteroatoms also include phosphorus,and selenium. As used herein, the term “cycloheteroalkyl” includingheterocyclyl and heterocycle, includes a chain of atoms that includesboth carbon and at least one heteroatom, such as heteroalkyl, and isoptionally branched, where at least a portion of the chain is cyclic.Illustrative heteroatoms include nitrogen, oxygen, and sulfur. Incertain variations, illustrative heteroatoms also include phosphorus,and selenium. Illustrative cycloheteroalkyl include, but are not limitedto, tetrahydrofuryl, pyrrolidinyl, tetrahydropyranyl, piperidinyl,morpholinyl, piperazinyl, homopiperazinyl, quinuclidinyl, and the like.

As used herein, the term “aryl” includes monocyclic and polycyclicaromatic carbocyclic groups, each of which may be optionallysubstituted. Illustrative aromatic carbocyclic groups described hereininclude, but are not limited to, phenyl, naphthyl, and the like. As usedherein, the term “heteroaryl” includes aromatic heterocyclic groups,each of which may be optionally substituted. Illustrative aromaticheterocyclic groups include, but are not limited to, pyridinyl,pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, quinolinyl, quinazolinyl,quinoxalinyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl,isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl,benzimidazolyl, benzoxazolyl, benzthiazolyl, benzisoxazolyl,benzisothiazolyl, and the like.

As used herein, the term “amino” includes the group NH₂, alkylamino, anddialkylamino, where the two alkyl groups in dialkylamino may be the sameor different, i.e. alkylalkylamino. Illustratively, amino includesmethylamino, ethylamino, dimethylamino, methylethylamino, and the like.In addition, it is to be understood that when amino modifies or ismodified by another term, such as aminoalkyl, or acylamino, the abovevariations of the term amino are included therein. Illustratively,aminoalkyl includes H₂N-alkyl, methylaminoalkyl, ethylaminoalkyl,dimethylaminoalkyl, methylethylaminoalkyl, and the like. Illustratively,acylamino includes acylmethylamino, acylethylamino, and the like.

As used herein, the term “amino and derivatives thereof” includes aminoas described herein, and alkylamino, alkenylamino, alkynylamino,heteroalkylamino, heteroalkenylamino, heteroalkynylamino,cycloalkylamino, cycloalkenylamino, cycloheteroalkylamino,cycloheteroalkenylamino, arylamino, arylalkylamino, arylalkenylamino,arylalkynylamino, heteroarylamino, heteroarylalkylamino,heteroarylalkenylamino, heteroarylalkynylamino, acylamino, and the like,each of which is optionally substituted. The term “amino derivative”also includes urea, carbamate, and the like.

As used herein, the term “hydroxy and derivatives thereof” includes OH,and alkyloxy, alkenyloxy, alkynyloxy, heteroalkyloxy, heteroalkenyloxy,heteroalkynyloxy, cycloalkyloxy, cycloalkenyloxy, cycloheteroalkyloxy,cycloheteroalkenyloxy, aryloxy, arylalkyloxy, arylalkenyloxy,arylalkynyloxy, heteroaryloxy, heteroarylalkyloxy, heteroarylalkenyloxy,heteroarylalkynyloxy, acyloxy, and the like, each of which is optionallysubstituted. The term “hydroxy derivative” also includes carbamate, andthe like.

As used herein, the term “thio and derivatives thereof” includes SH, andalkylthio, alkenylthio, alkynylthio, heteroalkylthio, heteroalkenylthio,heteroalkynylthio, cycloalkylthio, cycloalkenylthio,cycloheteroalkylthio, cycloheteroalkenylthio, arylthio, arylalkylthio,arylalkenylthio, arylalkynylthio, heteroarylthio, heteroarylalkylthio,heteroarylalkenylthio, heteroarylalkynylthio, acylthio, and the like,each of which is optionally substituted. The term “thio derivative” alsoincludes thiocarbamate, and the like.

As used herein, the term “acyl” includes formyl, and alkylcarbonyl,alkenylcarbonyl, alkynylcarbonyl, heteroalkylcarbonyl,heteroalkenylcarbonyl, heteroalkynylcarbonyl, cycloalkylcarbonyl,cycloalkenylcarbonyl, cycloheteroalkylcarbonyl,cycloheteroalkenylcarbonyl, arylcarbonyl, arylalkylcarbonyl,arylalkenylcarbonyl, arylalkynylcarbonyl, heteroarylcarbonyl,heteroarylalkylcarbonyl, heteroarylalkenylcarbonyl,heteroarylalkynylcarbonyl, acylcarbonyl, and the like, each of which isoptionally substituted.

As used herein, the term “carboxylic acid and derivatives thereof”includes the group CO₂H and salts thereof, and esters and amidesthereof, and CN.

The term “optionally substituted” as used herein includes thereplacement of hydrogen atoms with other functional groups on theradical that is optionally substituted. Such other functional groupsillustratively include, but are not limited to, amino, hydroxyl, halo,thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonicacids and derivatives thereof, carboxylic acids and derivatives thereof,and the like. Illustratively, any of amino, hydroxyl, thiol, alkyl,haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl,heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid isoptionally substituted.

As used herein, the terms “optionally substituted aryl” and “optionallysubstituted heteroaryl” include the replacement of hydrogen atoms withother functional groups on the aryl or heteroaryl that is optionallysubstituted. Such other functional groups illustratively include, butare not limited to, amino, hydroxy, halo, thio, alkyl, haloalkyl,heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl,heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic acids andderivatives thereof, carboxylic acids and derivatives thereof, and thelike. Illustratively, any of amino, hydroxy, thio, alkyl, haloalkyl,heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl,heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid isoptionally substituted.

Illustrative substituents include, but are not limited to, a radical—(CH₂)_(x)Z^(x), where x is an integer from 0-6 and Z^(x) is selectedfrom halogen, hydroxy, alkanoyloxy, including C₁-C₆ alkanoyloxy,optionally substituted aroyloxy, alkyl, including C₁-C₆ alkyl, alkoxy,including C₁-C₆ alkoxy, cycloalkyl, including C₃-C₈ cycloalkyl,cycloalkoxy, including C₃-C₈ cycloalkoxy, alkenyl, including C₂-C₆alkenyl, alkynyl, including C₂-C₆ alkynyl, haloalkyl, including C₁-C₆haloalkyl, haloalkoxy, including C₁-C₆ haloalkoxy, halocycloalkyl,including C₃-C₈ halocycloalkyl, halocycloalkoxy, including C₃-C₈halocycloalkoxy, amino, C₁-C₆ alkylamino, (C₁-C₆ alkyl)(C₁-C₆alkyl)amino, alkylcarbonylamino, N—(C₁-C₆ alkyl)alkylcarbonylamino,aminoalkyl, C₁-C₆ alkylaminoalkyl, (C₁-C₆ alkyl)(C₁-C₆ alkyl)aminoalkyl,alkylcarbonylaminoalkyl, N—(C₁-C₆ alkyl)alkylcarbonylaminoalkyl, cyano,and nitro; or Z^(x) is selected from —CO₂R⁴ and —CONR⁵R⁶, where R⁴, R⁵,and R⁶ are each independently selected in each occurrence from hydrogen,C₁-C₆ alkyl, aryl-C₁-C₆ alkyl, and heteroaryl-C₁-C₆ alkyl.

The term “prodrug” as used herein generally refers to any compound thatwhen administered to a biological system generates a biologically activecompound as a result of one or more spontaneous chemical reaction(s),enzyme-catalyzed chemical reaction(s), and/or metabolic chemicalreaction(s), or a combination thereof. In vivo, the prodrug is typicallyacted upon by an enzyme (such as esterases, amidases, phosphatases, andthe like), simple biological chemistry, or other process in vivo toliberate or regenerate the more pharmacologically active drug. Thisactivation may occur through the action of an endogenous host enzyme ora non-endogenous enzyme that is administered to the host preceding,following, or during administration of the prodrug. Additional detailsof prodrug use are described in U.S. Pat. No. 5,627,165; and Pathalk etal., Enzymic protecting group techniques in organic synthesis,Stereosel. Biocatal. 775-797 (2000). It is appreciated that the prodrugis advantageously converted to the original drug as soon as the goal,such as targeted delivery, safety, stability, and the like is achieved,followed by the subsequent rapid elimination of the released remains ofthe group forming the prodrug.

Prodrugs may be prepared from the compounds described herein byattaching groups that ultimately cleave in vivo to one or morefunctional groups present on the compound, such as —OH—, —SH, —CO₂H,—NR₂. Illustrative prodrugs include but are not limited to carboxylateesters where the group is alkyl, aryl, aralkyl, acyloxyalkyl,alkoxycarbonyloxyalkyl as well as esters of hydroxyl, thiol and amineswhere the group attached is an acyl group, an alkoxycarbonyl,aminocarbonyl, phosphate or sulfate. Illustrative esters, also referredto as active esters, include but are not limited to 1-indanyl,N-oxysuccinimide; acyloxyalkyl groups such as acetoxymethyl,pivaloyloxymethyl, β-acetoxyethyl, β-pivaloyloxyethyl,1-(cyclohexylcarbonyloxy)prop-1-yl, (1-aminoethyl)carbonyloxymethyl, andthe like; alkoxycarbonyloxyalkyl groups, such asethoxycarbonyloxymethyl, α-ethoxycarbonyloxyethyl,β-ethoxycarbonyloxyethyl, and the like; dialkylaminoalkyl groups,including di-lower alkylamino alkyl groups, such as dimethylaminomethyl,dimethylaminoethyl, diethylaminomethyl, diethylaminoethyl, and the like;2-(alkoxycarbonyl)-2-alkenyl groups such as2-(isobutoxycarbonyl)pent-2-enyl, 2-(ethoxycarbonyl)but-2-enyl, and thelike; and lactone groups such as phthalidyl, dimethoxyphthalidyl, andthe like.

Further illustrative prodrugs contain a chemical moiety, such as anamide or phosphorus group functioning to increase solubility and/orstability of the compounds described herein. Further illustrativeprodrugs for amino groups include, but are not limited to,(C₃-C₂₀)alkanoyl; halo-(C₃-C₂₀)alkanoyl; (C₃-C₂₀)alkenoyl;(C₄-C₇)cycloalkanoyl; (C₃-C₆)-cycloalkyl(C₂-C₁₆)alkanoyl; optionallysubstituted aroyl, such as unsubstituted aroyl or aroyl substituted by 1to 3 substituents selected from the group consisting of halogen, cyano,trifluoromethanesulphonyloxy, (C₁-C₃)alkyl and (C₁-C₃)alkoxy, each ofwhich is optionally further substituted with one or more of 1 to 3halogen atoms; optionally substituted aryl(C₂-C₁₆)alkanoyl, such as thearyl radical being unsubstituted or substituted by 1 to 3 substituentsselected from the group consisting of halogen, (C₁-C₃)alkyl and(C₁-C₃)alkoxy, each of which is optionally further substituted with 1 to3 halogen atoms; and optionally substituted heteroarylalkanoyl havingone to three heteroatoms selected from O, S and N in the heteroarylmoiety and 2 to 10 carbon atoms in the alkanoyl moiety, such as theheteroaryl radical being unsubstituted or substituted by 1 to 3substituents selected from the group consisting of halogen, cyano,trifluoromethanesulphonyloxy, (C₁-C₃)alkyl, and (C₁-C₃)alkoxy, each ofwhich is optionally further substituted with 1 to 3 halogen atoms. Thegroups illustrated are exemplary, not exhaustive, and may be prepared byconventional processes.

It is understood that the prodrugs themselves may not possesssignificant biological activity, but instead undergo one or morespontaneous chemical reaction(s), enzyme-catalyzed chemical reaction(s),and/or metabolic chemical reaction(s), or a combination thereof afteradministration in vivo to produce the compound described herein that isbiologically active or is a precursor of the biologically activecompound. However, it is appreciated that in some cases, the prodrug isbiologically active. It is also appreciated that prodrugs may oftenserves to improve drug efficacy or safety through improved oralbioavailability, pharmacodynamic half-life, and the like. Prodrugs alsorefer to derivatives of the compounds described herein that includegroups that simply mask undesirable drug properties or improve drugdelivery. For example, one or more compounds described herein mayexhibit an undesirable property that is advantageously blocked orminimized may become pharmacological, pharmaceutical, or pharmacokineticbarriers in clinical drug application, such as low oral drug absorption,lack of site specificity, chemical instability, toxicity, and poorpatient acceptance (bad taste, odor, pain at injection site, and thelike), and others. It is appreciated herein that a prodrug, or otherstrategy using reversible derivatives, can be useful in the optimizationof the clinical application of a drug.

As used herein, the term “treating”, “contacting” or “reacting” whenreferring to a chemical reaction means to add or mix two or morereagents under appropriate conditions to produce the indicated and/orthe desired product. It should be appreciated that the reaction whichproduces the indicated and/or the desired product may not necessarilyresult directly from the combination of two reagents which wereinitially added, i.e., there may be one or more intermediates which areproduced in the mixture which ultimately leads to the formation of theindicated and/or the desired product.

As used herein, the term “composition” generally refers to any productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationsof the specified ingredients in the specified amounts. It is to beunderstood that the compositions described herein may be prepared fromisolated compounds described herein or from salts, solutions, hydrates,solvates, and other forms of the compounds described herein. It is alsoto be understood that the compositions may be prepared from variousamorphous, non-amorphous, partially crystalline, crystalline, and/orother morphological forms of the compounds described herein. It is alsoto be understood that the compositions may be prepared from varioushydrates and/or solvates of the compounds described herein. Accordingly,such pharmaceutical compositions that recite compounds described hereinare to be understood to include each of, or any combination of, thevarious morphological forms and/or solvate or hydrate forms of thecompounds described herein. Illustratively, compositions may include oneor more carriers, diluents, and/or excipients. The compounds describedherein, or compositions containing them, may be formulated in atherapeutically effective amount in any conventional dosage formsappropriate for the methods described herein. The compounds describedherein, or compositions containing them, including such formulations,may be administered by a wide variety of conventional routes for themethods described herein, and in a wide variety of dosage formats,utilizing known procedures (see generally, Remington: The Science andPractice of Pharmacy, (21^(st) ed., 2005)).

EXAMPLES

Example Synthesis of Dipeptide 3

4.9 g of dipeptide 1 (11.6 mmol) was dissolved in 60 mL dichloromethane,imidazole (0.87 g, 12.7 mmol) was added to the resulting solution at 0°C. The reaction mixture was warmed slightly to dissolve all solids andre-cooled to 0° C. TESCl (2.02 mL, 12.1 mmol) was added drop-wise at 0°C., the reaction mixture was stirred under argon and warmed to roomtemperature over 2 h. TLC (3:1 hexanes/EtOAc) showed completeconversion. The reaction was filtered to remove the imidazole HCl salt,extracted with de-ionized water, and the aqueous phase was back-washedwith dichloromethane, the combined organic phase was washed with brine,dried over Na₂SO₄, filtered to remove the Na₂SO₄, concentrated underreduced pressure, co-evaporated with toluene and dried under high-vacuumovernight to give 6.4 g of crude product 2 (vs 5.9 g of theoreticalyield).

The crude product 2 was co-evaporated with toluene again and usedwithout further purification. TES protected dipeptide was dissolved in38 mL THF (anhydrous, inhibitor-free) and cooled to −45° C. and stirredfor 15 minutes before adding KHMDS (0.5 M in toluene, 25.5 mL, 12.8mmol, 1.1 equiv) drop-wise. After the addition of KHMDS was complete,the reaction mixture was stirred at −45° C. for 15 minutes, andchloromethyl butyrate (1.8 mL, 1.2 equiv, 14 mmol) was added. Thereaction mixture changed from light yellow to a blueish color. TLC (20%EtOAc/petroleum ether) showed the majority of starting material wasconverted. LC-MS showed about 7% starting material left. The reactionwas quenched by adding 3 mL MeOH, the mixture was warmed to roomtemperature and concentrated under reduced pressure to an oily residue.The residue was dissolved in petroleum ether and passed through shortsilica plug to remove the potassium salt. The plug was washed with 13%EtOAc/petroleum ether, and the collected eluates were combined andconcentrated under reduced pressure. The crude alkylated product waspassed through an additional silica plug (product/silica=1:50) andeluted with 13% EtOAc/petroleum ether to remove residual startingmaterial to give 5.7 g of product 3 (two steps, yield 76%)

Example Synthesis of Tripeptide 4

Alkylated dipeptide 3 (4.3 g, 7.0 mmol), N-methyl pipecolinate (MEP)(4.0 g, 28.0 mmol, 4 equiv) and pentafluorophenol (5.7 g, 30.8 mmol. 4.4equiv) were added to a flask. N-methylpyrrolidone (NMP, 86 mL) was addedto the mixture. To the mixture was added diisopropylcarbodiimide (DIC,4.77 mL, 30.8 mmol, 4.4 equiv) was added to the mixture. The mixture wasstirred at room temperature for 1 h. Pd/C (10%, dry, 1.7 g) was added.The flask was shaken under hydrogen (30-35 psi) for 5 hours. Thereaction mixture was analyzed by HPLC. The starting material was foundto be less than 3%. The mixture was filtered through diatomaceous earth.The diatomaceous earth was extracted with 200 mL ethyl acetate. Thefiltrate and the ethyl acetate extract were combined and transferred toa separatory funnel and washed with 1% NaHCO₃/10% NaCl solution (200mL×4). The organic layer was isolated and evaporated on a rotaryevaporator under reduced pressure. The crude product was dissolved in 40mL of MeOH/H₂O (3:1). The crude product solution was loaded onto aBiotage C18 column (Flash 65i, 350 g, 450 mL, 65×200 mm) and eluted withbuffer A [10 mM NH₄OAc/ACN (1:1)] and B (ACN, acetonitrile). Thefractions were collected and organic solvent was removed by evaporatingon a rotary evaporator. 100 mL of 10% NaCl solution and 100 mL of methyltert-butyl ether (MTBE) were added to the flask and the mixture wastransferred to a separatory funnel. The organic layer was isolated anddried over anhydrous Na₂SO₄, filtered and evaporated on a rotaryevaporator to dryness. 2.5 g of tripeptide intermediate 4 was obtained(yield 50%).

Example Large Scale Synthesis of Dipeptide 3

10.2 g of dipeptide 1 (25.6 mmol) was dissolved in 130 mLdichloromethane, imidazole (1.9 g, 28.1 mmol) was added to the resultingsolution at 0° C. The reaction mixture was warmed slightly to dissolveall solids and re-cooled to 0° C. TESCl (4.5 mL, 26.8 mmol) was addeddrop-wise at 0° C., the reaction mixture was stirred under argon andwarmed to room temperature over 2 h. TLC (3:1 hexanes/EtOAc) showedcomplete conversion. The reaction was filtered to remove the imidazoleHCl salt, extracted with de-ionized water, and the aqueous phase wasback-washed with dichloromethane, the combined organic phase was washedwith brine, dried over Na₂SO₄, filtered to remove the Na₂SO₄,concentrated under reduced pressure, co-evaporated with toluene anddried under high-vacuum overnight to give 12.2 g of product 2.

The crude product 2 was co-evaporated with toluene again and usedwithout further purification. TES protected dipeptide was dissolved in80 mL THF (anhydrous, inhibitor-free) and cooled to −45° C. and stirredfor 15 minutes before adding KHMDS (0.5 M in toluene, 50 mL, 25.0 mmol,1.05 equiv) drop-wise. After the addition of KHMDS was complete, thereaction mixture was stirred at −45° C. for 15 minutes, and chloromethylbutyrate (3.6 mL, 1.2 equiv, 28.3 mmol) was added. The reaction mixturechanged from light yellow to a blueish color. TLC (20% EtOAc/petroleumether) showed the reaction was complete. The reaction was quenched byadding 20 mL MeOH, the mixture was warmed to room temperature andconcentrated under reduced pressure to an oily residue. The residue wasdissolved in petroleum ether and passed through short silica plug toremove the potassium salt. The plug was washed with 13% EtOAc/petroleumether, and the collected eluents were combined and concentrated underreduced pressure to give 12.1 g of product 3 (two steps, yield 76%)

Example Large Scale Synthesis of Tripeptide 4

Alkylated dipeptide 3 (7.6 g, 12.4 mmol), N-methyl pipecolinate (MEP)(7.0 g, 48.9 mmol, 4 equiv) and pentafluorophenol (10.0 g, 54.3 mmol.4.4 equiv) were added to a flask. N-methylpyrrolidone (NMP, 152 mL) wasadded to the mixture. To the mixture was added diisopropylcarbodiimide(DIC, 8.43 mL, 54.4 mmol, 4.4 equiv) was added to the mixture. Themixture was stirred at room temperature for 1 h. Pd/C (10%, dry, 3.0 g)was added. The flask was shaken under hydrogen (30-35 psi) for 5 hours.The reaction mixture was analyzed by HPLC. The reaction was complete.The mixture was filtered through celite. The celite was washed with 500mL ethyl acetate. The solutions were combined and transferred to aseparatory funnel and washed with 1% NaHCO₃/10% NaCl solution (250mL×4). The organic layer was isolated and evaporated on a rotaryevaporator under reduced pressure. The crude product was dissolved indichloromethane and the urea was filtered. The crude product solutionwas loaded onto a Teledyne Redisep Silica Column (330 g) and purifiedwith EtOAc/petroleum ether on CombiFlash flash chromatography system.The fractions were collected and organic solvent was removed byevaporating to give 5.0 g of the tripeptide (61%). NMR and mass spectraldata were consistent with those measured for the Example

Also described herein, is the conversion of 4 to 10 (R remains Me) bycontacting 4 with TFA and an alcohol. In some illustrative examples ofcompound 10, R is allyl, or CH₂(CH₂)nCH₃, where n is 1, 2, 3, 4, 5, or6.

Example

Compound 4 (50 mg, 0.07 mmol) in allyl alcohol (5 mL) was treated withdi-n-butyltin oxide (1.75 mg, 0.007 mmol, 10% mol). The reaction mixturewas heated to reflux for 22 hrs till the reaction was complete. Thereaction was concentrated and purified with HPLC in 10-100% ACN/NH₃HCO₃buffer (pH7.0) to give the title compound (32.4 mg, yield 65%). LCMS:[M+H]⁺ m/z=707.73. ¹H NMR (CD₃OD, δ in ppm): 8.35 (s, 1H), 6.01 (m, 2H);5.2-5.5 (m, 3H), 5.14 (d, J=10.26 Hz, 1H), 5.04 (d, J=5.87 Hz, 1H), 4.88(s, 3H), 4.82 (d, J=5.5 Hz, 2H), 4.70 (d, J=8.79 Hz, 1H), 4.50 (d,J=10.26 Hz, 1H), 4.42 (b, 1H), 4.06 (s, 2H), 2.92 (d, J=11.36 Hz, 1H),2.55 (d, J=9.17 Hz, 1H), 1.95-2.20 (m, 7H), 1.45-1.82 (m, 7H), 1.22 (m,2H), 0.82-1.00 (m, 17H), 0.77 (d, J=6.23 Hz, 3H), 0.59-0.70 (m, 6H); ¹³CNMR (CD₃OD, δ in ppm): 176.97, 175.08, 174.09, 160.95, 146.02, 134.13,132.05, 127.94, 117.38, 116.37, 73.85, 70.32, 69.14, 68.40, 65.34,56.89, 55.20, 53.55, 43.35, 40.37, 36.38, 31.59, 30.15, 24.80, 24.27,22.93, 19.09, 18.71, 15.31, 9.52, 5.77, 4.41.

Example

Compound 10a (15.3 mg, 0.02 mmol) was subjected to hydrolysis withLiOH.H₂O (0.99 mg, 0.024 mmol) in 4:1 THF/H₂O (2.5 mL) for 19 hrs atroom temperature (rt). The reaction was purified with HPLC in 10-100%ACN/NH₃HCO₃ buffer (pH7.0) to provide compound 11a (9.2 mkg, yield 83%).LCMS: [M+H]⁺ m/z=553.55. ¹H NMR (CD₃OD, δ in ppm): 7.94 (s, 1H), 6.00(m, 1H), 5.1-5.4 (m, 3H), 4.68 (d, J=9.09 Hz, 2H), 4.10 (d, J=3.81 Hz,2H), 2.80 (b, 1H), 2.56 (s, 2H), 1.4-2.2 (m, 11H), 1.20 (m, 1H),0.80-0.99 (m, 13H); ¹³C NMR (CD₃OD, δ in ppm): 17.90, 167.53, 153.18,134.05, 123.09, 116.53, 68.63, 67.25, 54.85, 54.44, 42.10, 37.75, 36.53,30.60, 29.13, 24.26, 23.25, 21.37, 20.32, 19.53, 14.72, 9.51.

Example

To compound 11a (9.2 mg, 0.017 mmol) in pyridine (1 mL) was added aceticanhydride (15.7 μL, 0.165 mmol) and a catalytic amount of4-dimethylamino pyridine (0.053 M in pyridine, 5 μL) at rt under argon.The reaction was stirred for 24 hrs. To the reaction mixture was added0.4 mL of dioxane/water (1:1) and stirred for 10 min, and then thesolvent was removed in vacuo. The residue was purified with HPLC in10-100% ACN/NH₃HCO₃ buffer (pH7.0) to provide the product 12a 10.4 mg(quantitative yield). LCMS: [M+H]⁺ m/z=595.59. ¹H NMR (CD₃OD, δ in ppm):7.96 (s, 1H), 5.8-6.0 (m, 2H), 5.33 (d, J=17.59 Hz, 1H), 5.19 (d,J=10.56 Hz, 1H), 4.71 (d, J=9.23 Hz, 2H), 4.05 (d, J=5.71 Hz, 2H), 3.30(m, 6H), 2.50 (b, 4H), 2.10 (s, 3H), 1.40-2.00 (m, 7H), 1.20 (m, 1H),0.80-1.02 (m, 11H); ¹³C NMR (CD₃OD, δ in ppm): 175.11, 170.44, 167.29,153.45, 133.92, 123.40, 116.79, 116.55, 68.62, 67.82, 67.11, 54.75,54.16, 42.39, 36.31, 36.12, 34.91, 30.55, 29.26, 24.09, 23.26, 21.25,20.24, 19.48, 19.20, 14.78, 9.56.

Example

Compound 12a (10.4 mg, 0.017 mmol) was dissolved in anhydrous methylenechloride (4 mL) and to this solution was added DCC-resin (2.3 mmol/g,0.038 g, 0.087 mmol) and followed by pentafluorophenol (PFP, 6.26 mg,0.034 mmol) at rt under argon. The reaction was stirred for 19 hrs atrt. The reaction mixture was filtered and the solution was concentrated.The residue was redissolved in dry DMF (4 mL). Then,(2S,4R)-4-amino-5-(4-hydroxyphenyl)-2-methylpentanoic acid (Tut acid)was added into the solution, followed by DIPEA (8.9 μL, 0.051 mmol).When completed, the reaction was concentrated in vacuo and the residuewas purified with HPLC. Product 13a was obtained (13.1 mg, 96% yield).LCMS: [M+H]⁺ m/z=800.88. ¹H NMR (CD₃OD, δ in ppm): 8.08 (s, 1H), 7.02(d, J=8.43 Hz, 2H), 6.68 (d, J=8.06 Hz, 2H), 5.99 (d, J=10.99 Hz, 1H),5.80 (m, 1H), 5.38 (d, J=9.53 Hz, 1H), 5.31 (d, J=17.23 Hz, 1H), 5.13(d, J=10.63 Hz, 1H), 4.66 (d, J=8.79 Hz, 1H), 4.55 (d, J=10.28 Hz, 1H),4.30 (b, 2H), 4.00 (b, 2H), 3.16 (b, 2H), 2.80 (d, J=5.86 Hz, 2H), 2.40(b, 4H), 2.10-2.30 (b, 2H), 1.40-1.90 (b, 6H), 1.23 (s, 3H), 1.17 (d,J=6.96 Hz, 3H), 1.05 (d, J=6.23 Hz, 2H), 0.94 (d, J=6.97 Hz, 2H), 0.90(d, J=7.70 Hz, 2H), 0.79 (d, J=6.6 Hz, 3H); ¹³C NMR (CD₃OD, δ in ppm):179.24, 174.88, 170.97, 170.43, 170.20, 161.29, 155.62, 149.30, 133.70,130.23, 128.44, 123.54, 116.41, 114.72, 69.92, 68.15, 67.87, 54.96,53.92, 49.27, 42.40, 39.62, 37.72, 36.91, 36.08, 35.29, 31.01, 29.51,29.33, 24.08, 23.72, 21.93, 19.40, 19.34, 18.89, 17.24, 15.00, 9.34.

Example

Compound 12a (26.4 mg, 0.044 mmol) was dissolved in anhydrous methylenechloride (5 mL) and to this solution was added DCC-resin (2.3 mmol/g,0.096 g, 0.22 mmol), followed by pentafluorophenol (PFP, 16.4 mg, 0.089mmol) at rt under argon. The reaction was stirred for 19 hrs at rt. Thereaction was filtered and concentrated and the residue was redissolvedin dry DMF (5 mL). 2-((3-nitropyridin-2-yl)disulfanyl)ethyl2-((2S,4R)-4-((tert-butoxycarbonyl)amino)-5-(4-hydroxyphenyl)-2-methylpentanoyl)hydrazinecarboxylate(40.0 mg, 0.067 mmol) was deprotected with TFA/DCM (1:1, 5 mL, 1 drop ofTIPS as scavenger) at rt for 1 hr. The solvent was removed under reducedpressure, 5 mL more of DCM was added, and then the solvent wasco-evaporated to dryness. The residue was dissolved in dry DMF (2 mL)and was added to the solution of PFP ester intermediate in DMF madeabove after the addition of DIPEA (23.2 μL, 0.13 mmol) at rt underargon. The reaction was stirred for 19 hrs and diluted with EtOAc (20mL). The organic phase was washed with water (5 mL×3) and brine. Theorganic layer was dried over anhydrous Na₂SO₄ and concentrated afterfiltration to give the crude product 15a (52.8 mg), which could be usedfor conjugation with folate. LCMS: [M+H]⁺ m/z=1072.92.

Example

Compound 4 (75.9 mg, 0.11 mmol) in n-butanol (4 mL) was treated withn-Bu₂SnO (2.12 mg, 0.0085 mmol, 8.0 mol %) at rt and the reaction washeated to 100° C. for 2 days. The solvent was reduced to a minimum andthe product was purified with CombiFlash (Teledyne Redisep Silicacolumn, eluted with 0 to 15% of MeOH/DCM) to give 44.0 mg (56%) ofintermediate 10b. LCMS: [M+H]⁺ m/z=739.61. ¹H NMR (CDCl₃, δ in ppm):8.07 (s, 1H), 7.02 (d, J=9.68 Hz, 1H), 5.27 (d, J=9.67 Hz, 1H), 5.02(dd, J=8.36, 2.64 Hz, 1H), 4.69 (t, J=9.23 Hz, 4.20-4.40 (m, 4H), 3.47(td, J=6.6, 1.76 Hz, 2H), 2.88 (d, J=11.44 Hz, 1H), 2.46 (dd, J=10.55,3.08 Hz, 2H), 1.90-2.24 (m, 8H), 1.10-1.79 (m, 18H), 0.80-1.00 (m, 19H),0.58-0.78 (m, 6H).

Example

The same procedure as compound 11a was followed. 11b (11.7 mg, 35%) wasobtained from intermediate 10b (44.0 mg). LCMS: [M+H]⁺ m/z=569.51. ¹HNMR (CDCl₃ drops of CD₃OD, δ in ppm) 8.00 (s, 1H), 5.23 (b, 1H), 4.80(b, 1H), 4.58 (d, J=8.80 Hz, 1H), 4.42 (b, 1H), 3.45 (t, J=6.38 Hz, 1H),3.33 (b, 3H), 2.15-2.40 (m, 3H), 1.80-2.10 (m, 2H), 1.40-1.79 (m, 4H),1.04-1.38 (m, 3H), 0.60-1.02 (m, 9H).

Example

In a 10 mL round bottom flask, 11b (11.7 mg, 0.021 mmol) and aceticanhydride (20 μL, 0.212 mmol) were dissolved in pyridine (1 mL). To thissolution was added a catalytic amount of dimethylaminopyridine (1 mg,0.008 mmol). This solution was stirred at room temperature for 16 hunder Argon. LCMS (10-100% ACN, 50 mM NH₄HCO₃ pH7) indicated all of thestarting material had been consumed and product had been formed. To theflask was added a 1:1 mixture of 1,4-dioxane and water (0.4 mL) and thesolution was stirred for 10 min to hydrolyze any potential diacetateside product. The reaction mixture was concentrated under reducedpressure, then purified by preparative HPLC (10-100% ACN, 50 mM NH₄HCO₃pH7) to yield 12b (9.6 mg, 76%). LCMS: [M+H]⁺=611.53. ¹H NMR (CDCl₃ w/2drops CD₃OD): 7.97 (s, 1H) 5.83 (d, J=9.9 Hz, 1H) 5.28 (s, 1H) 4.58 (d,J=9.0 Hz, 1H) 4.24 (d, J=9.3 Hz, 2H) 3.42 (m, 3H) 2.60-2.95 (br, 7H)2.20-2.58 (br, 6H) 1.76-2.20 (br, ¹H) 1.40-1.56 (br, 12H) 1.02-1.20 (br,12H) 0.40-1.10 (br, 27H) 0.04 (s, 8H). ¹³C NMR: 175.04, 170.53, 67.78,53.74, 44.33, 36.79, 35.64, 31.69, 29.89, 24.86, 20.96, 20.49, 19.52,15.95, 13.99, 10.65, 1.21

Example

In a 25 mL round bottom flask, 12b (9.6 mg, 0.016 mmol) andpentafluorophenol (28.2 mg, 0.153 mmol) were dissolved in drydichloromethane (5 mL). N-cyclohexylcarbodiimide, N′-methyl polystyrene(33.4 mg, 2.3 mmol/g, 0.077 mmol) was added and the reaction mixture wasstirred at room temperature for 16 h under Argon. LCMS (10-100% ACN, 50mM NH₄HCO₃ pH7) indicated all of the starting material had been consumedand activated intermediate had been formed. The reaction mixture wasfiltered and concentrated under reduced pressure, and the residue wasdissolved in a solution of N,N-dimethylformamide (2 mL) andN,N-diisopropylethylamine (8 μL, 0.046 mmol). PFP ester intermediate(6.0 mg, 0.023 mmol) was added and the reaction mixture was stirred atroom temperature for 2 h under argon. LCMS (10-100% ACN, 50 mM NH₄HCO₃pH7) indicated all of the activated intermediate had been consumed andproduct had been formed. The reaction mixture was purified bypreparative HPLC (10-100% ACN, 50 mM NH₄HCO₃ pH7) to yield 13b (4.7 mg,37%). LCMS: [M+H]⁺ m/z=816.71. ¹H NMR (CDCl₃, δ in ppm): 8.04 (s, 1H)7.05 (d, J=8.4 Hz, 2H) 6.80 (d, J=8.4 Hz, 2H) 5.90 (m, 1H) 5.38 (d,J=10.2 Hz, 1H) 4.63 (t, J=9.3 Hz, 1H) 4.38 (br, 1H) 4.27 (d, J=9.9 Hz,1H) 3.48 (m, 1H) 3.34 (m, 2H) 2.86 (m, 6H) 2.56 (m, 3H) 2.23 (s, 3H)2.16 (s, 3H) 1.22-2.10 (br, 16H) 1.12 (d, J=6.9 Hz, 3H) 1.03 (d, J=6.6Hz, 3H) 0.88 (m, 14H). ¹³C NMR: 174.90, 170.44, 161.73, 155.52, 149.37,130.77, 128.56, 124.33, 115.91, 70.40, 69.69, 67.62, 55.45, 53.70,49.25, 44.61, 40.40, 36.94, 36.69, 35.93, 31.77, 31.16, 30.06, 24.94,23.14, 21.08, 20.74, 20.20, 19.55, 17.78, 16.08, 14.05, 10.70

Example

Compound 4 (73.9 mg, 0.10 mmol) in n-pentanol (4 mL) was treated withn-Bu₂SnO (2.10 mg, 0.0083 mmol, 8.0 mol %) at rt and the reaction washeated to 100° C. for 2 days. The solvent was reduced to a minimum andthe product was purified with CombiFlash (Teledyne Redisep Silicacolumn, eluted with 0 to 15% of MeOH/DCM) to give 51.2 mg (64%) ofintermediate 10b. LCMS: [M+H]⁺ m/z=767.64. ¹H NMR (CDCl₃, δ in ppm):8.07 (m, 1H), 7.06 (t, J=9.23 Hz, 1H), 5.95 (d, J=12.3 Hz, 1H), 5.43 (d,J=12.32 Hz, 1H), 5.26 (d, J=9.68 Hz, 1H), 5.03 (dd, J=8.36, 2.64 Hz,1H), 4.93 (dd, J=8.36, 6.24 Hz, 1H), 4.71 (dd, J=15.83, 8.80 Hz, 1H),4.20-4.33 (m, 3H), 3.46 (m, 1H), 2.88 (d, J=11.43 Hz, 1H), 2.30-2.60 (m,2H), 2.20 (s, 2H), 1.95-2.18 (m, 3H), 1.50-1.80 (m, 6H), 1.10-1.44 (m,6H), 0.80-1.04 (m, 13H), 0.50-0.77 (m, 6H).

Example

The same procedure as for compound 11a was followed, intermediate 11c(14.9 mg, 38%) was obtained from 10c (51.2 mg). LCMS: [M+H]⁺ m/z=583.56.¹H NMR (CD₃OD, δ in ppm): 7.97 (s, 1H), 5.27 (d, J=9.67 Hz, 1H), 4.67(d, J=9.23 Hz, 1H), 4.58 (d, J=9.68 Hz, 1H), 3.53 (m, 3H), 2.80 (b, 1H),2.58 (b, 4H), 1.48-2.18 (m, 13H), 1.10-1.42 (m, 6H), 0.70-1.08 (m, 18H).

Example

In a 10 mL round bottom flask, 11c (14.9 mg, 0.026 mmol) and aceticanhydride (20 μL, 0.212 mmol) were dissolved in pyridine (1 mL). Thissolution was added a catalytic amount of dimethylaminopyridine (1 mg,0.008 mmol). This solution was stirred at room temperature for 16 hunder argon. LCMS (10-100% ACN, 50 mM NH₄HCO₃ pH7) indicated all of thestarting material had been consumed and product had been formed. To theflask was added a 1:1 mixture of 1,4-dioxane and water (0.4 mL) and thesolution was stirred for 10 min to hydrolyze any potential diacetateside product. The reaction mixture was concentrated under reducedpressure, then purified by preparative HPLC (10-100% ACN, 50 mM NH₄HCO₃pH7) to yield 12c (4.8 mg, 30%). LCMS: [M+H]⁺ m/z=625.58. ¹H NMR (CDCl₃w/2 drops CD₃OD) 7.98 (s, 1H) 5.82 (d, J=10.8 Hz, 1H) 5.26 (s, 1H) 4.57(d, J=8.4 Hz, 1H) 4.23 (d, J=8.4 Hz, 2H) 3.42 (m, 3H) 2.60-2.92 (br, 8H)2.15-2.40 (br, 4H) 1.90-2.12 (m, 7H) 1.38-1.90 (br, 14H) 1.00-1.38 (br,13H) 0.50-1.00 (br, 22H), 0.03 (s, 13H). ¹³C NMR: 175.15, 150.56,125.47, 69.55, 68.09, 55.33, 53.71, 44.59, 36.77, 35.74, 31.34, 30.19,29.86, 29.32, 28.51, 24.84, 22.85, 22.55, 20.86, 20.40, 19.91, 15.94,14.10, 10.63, 1.17

Example

In a 25 mL round bottom flask, 12c (4.8 mg, 0.008 mmol) andpentafluorophenol (14.1 mg, 0.077 mmol) were dissolved in drydichloromethane (5 mL). N-cyclohexylcarbodiimide, N-methyl polystyrene(16.7 mg, 2.3 mmol/g, 0.038 mmol) was added and the reaction mixture wasstirred at room temperature for 16 h under Argon. LC-MS (10-100% ACN, 50mM NH₄HCO₃ pH7) indicated all of the starting material had been consumedand activated intermediate had been formed. The reaction mixture wasfiltered and concentrated under reduced pressure, and the residue wasdissolved in a solution of N,N-dimethylformamide (2 mL) andN,N-diisopropylethylamine (4 μL, 0.023 mmol). PFP ester intermediate(3.0 mg, 0.012 mmol) was added and the reaction mixture was stirred atroom temperature for 2 h under Argon. LC-MS (10-100% ACN, 50 mM NH₄HCO₃pH7) indicated all of the activated intermediate had been consumed andproduct had been formed. The reaction mixture was purified bypreparative HPLC (10-100% ACN, 50 mM NH₄HCO₃ pH7) to yield 13c (1.1 mg,17%). LCMS: [M+H]⁺ m/z=830.76. ¹H NMR (CDCl₃ w/2 drops CD₃OD): 8.00 (s,1H) 7.01 (d, J=8.7 Hz, 2H) 6.74 (d, J=8.4 Hz, 2H) 5.89 (d, J=12.6 Hz,1H) 5.25 (d, J=9.0 Hz, 1H) 4.55 (d, J=8.7 Hz, 1H) 4.30 (m, 3H) 3.39 (m,3H) 3.21 (m, 2H) 2.81 (m, 3H) 2.04-2.60 (br, 45H) 1.76-2.04 (m, 5H)1.34-1.76 (br, 9H) 1.20 (m, 6H) 1.12 (d, J=7.2 Hz, 4H) 1.01 (d, J=6.3Hz, 3H) 0.89 (t, J=7.1 Hz, 6H) 0.78 (m, 6H)

Example

In a 5 mL round bottom flask, 14 (10.0 mg, 0.009 mmol) was dissolved ina solution of trifluoroacetic acid (125 μL, 1.632 mmol) anddichloromethane (0.5 mL) and stirred at room temperature for 1 hr underargon, then 1-butanol (200 μL, 2.186 mmol) added and reaction mixturestirred at room temperature for 30 min under argon. LCMS (10-100% ACN,50 mM NH₄HCO₃ pH7) indicated all of the starting material had beenconsumed and product had been formed. The reaction mixture was purifiedby preparative HPLC (10-100% ACN, 50 mM NH₄HCO₃ pH7) to yield 15b (3.2mg, 32%). LCMS: [M+H]⁺ m/z=1088.79. ¹H NMR (CDCl₃ w/2 drops CD₃OD): 8.86(s, 1H) 8.47 (d, J=8.0 Hz, 1H) 7.99 (s, 1H) 7.31 (d, J=9.5 Hz, 2H) 7.01(d, J=7.5 Hz, 2H) 6.73 (d, J=8.5 Hz, 2H) 5.94 (d, J=10.5 Hz, 1H) 5.34(d, J=10.0 Hz, 1H) 4.58 (m, 3H) 4.38 (t, J=6.0 Hz, 4H), 4.27 (d, J=10.0Hz, 2H) 3.37 (m, 2H) 3.18 (m, 2H) 3.09 (t, J=6.3 Hz, 3H) 2.70-2.90 (br,6H) 2.43 (dd, J=11.0 Hz, 3.0 Hz, 2H) 2.26-2.36 (br, 4H) 2.12-2.22 (br,10H) 2.02-2.12 (br, 2H) 1.86-2.02 (br, 11H) 1.69-1.80 (br, 6H) 1.54-1.69(br, 10H) 1.34-1.52 (br, 12H) 1.09-1.34 (br, 16H) 1.047 (dd, J=15.0 Hz,6.5 Hz, 19H) 0.88 (m, 19H) 0.75 (m, 17H). ¹³C NMR: 174.95, 174.59,170.64, 170.23, 161.92, 156.91, 156.06, 153.88, 149.00, 133.77, 130.79,123.92, 120.98, 115.53, 69.95, 69.61, 67.03, 63.82, 55.32, 53.21, 44.78,41.42, 40.40, 36.84, 36.38, 35.62, 35.22, 31.54, 31.40, 30.37, 24.99,24.66, 23.20, 20.68, 20.24, 19.56, 19.27, 17.69, 15.71, 13.72, 10.35

Example

In a 5 mL round bottom flask, 14 (10.0 mg, 0.009 mmol) was dissolved ina solution of trifluoroacetic acid (125 μL, 1.632 mmol) anddichloromethane (0.5 mL) and stirred at room temperature for 1 hr underargon, then 1-pentanol (200 μL, 1.840 mmol) added and reaction mixturestirred at room temperature for 30 min under argon. LC-MS (10-100% ACN,50 mM NH₄HCO₃ pH7) indicated all of the starting material had beenconsumed and product had been formed. The reaction mixture was purifiedby preparative HPLC (10-100% ACN, 50 mM NH₄HCO₃ pH7) to yield 15c (3.6mg, 36%). LCMS: [M+H]⁺ m/z=1102.77.

Example

In a 25 mL round bottom flask, 15b (3.2 mg, 0.003 mmol) was dissolved indimethylsulfoxide (2 mL). A solution of 16 (4.9 mg, 0.003 mmol) in 20mM, pH7, sodium phosphate buffer (2 mL) was added dropwise, stirring atroom temperature with argon bubbling for 30 min. LCMS (10-100% ACN, 50mM NH₄HCO₃ pH7) indicated all of the starting material had been consumedand product had been formed. The reaction mixture was purified bypreparative HPLC (10-100% ACN, 50 mM NH₄HCO₃ pH7) to yield 17b (4.3 mg,56%). LCMS: [M+H]⁺ m/z=1306.82. ¹H NMR (9:1 DMSO-d6:D₂O): 8.60 (s, 1H)8.14 (s, 1H) 7.59 (d, J=8.5 Hz, 2H) 6.94 (d, J=7.5 Hz, 2H) 6.60 (dd,J=13.3 Hz, 8.8 Hz, 3H) 5.77 (d, J=11.5 Hz, 1H) 5.20 (d, J=9.5 Hz, 1H)4.46 (m, 3H) 4.00-4.40 (br, 12H) 3.48-3.62 (br, 11H) 3.28-3.48 (br, 12H)3.10-3.28 (br, 4H) 2.80-3.08 (br, 7H) 2.60-3.80 (br, 3H) 2.48 (s, 1H)2.26-2.40 (br, 2H) 2.00-2.26 (br, 19H) 1.58-2.00 (br, 20H) 1.28-1.58(br, 8H) 1.18 (q, J=7.5 Hz, 3H) 0.84-1.10 (br, 8H) 0.75 (m, 9H) 0.60 (d,J=6.5 Hz, 3H). ¹³C NMR: 175.25, 174.93, 174.36, 173.59, 173.22, 172.76,172.70, 172.02, 171.85, 171.67, 170.85, 170.34, 169.68, 166.48, 161.94,160.67, 156.42, 155.80, 154.22, 150.98, 149.56, 149.21, 149.08, 130.64,129.17, 128.69, 128.08, 124.89, 122.00, 115.31, 111.84, 72.31, 72.23,71.82, 71.69, 69.84, 69.74, 68.21, 66.59, 63.52, 63.09, 55.04, 53.74,53.56, 53.23, 52.96, 52.48, 46.11, 43.63, 42.39, 37.43, 35.69, 35.41,35.19, 32.17,

Example

1.1 g of dipeptide 1 (2.77 mmole), was mixed with 53 mg (0.21 mmole,0.08 eq) of n-Bu₂SnO in 15 mL of benzyl alcohol and heated to 130° C.for 2½ hours, then 100° C. overnight. LC/MS showed no starting materialleft. The reaction mixture was loaded onto a 330 g of Combiflash column,purified with petroleum ether/EtOAc to give some clean fractions. Mixedfractions were repurified to give a combined yield of 0.67 g (51%) ofpure benzyl ester 18. LCMS: [M+H]⁺ m/z=474.46. ¹H NMR (CDCl₃, 6 in ppm):8.12 (s, 1H), 7.46-7.43 (m, 2H), 7.40-7.32 (m, 3H), 6.68 (d, J=9.6 Hz,1H), 5.41 (d, J=12.3 Hz, 1H), 5.36 (d, J=12.3 Hz, 1H), 5.24 (d, J=4.5Hz, 1H), 4.87 (m, 1H), 4.02-3.90 (m, 2H), 2.24-2.13 (m, 2H), 1.88-1.78(m, 2H), 1.42-1.30 (m, 2H), 1.07 (d, J=6.9 Hz, 3H), 0.97-0.90 (m, 9H).¹³C NMR (CDCl₃, 6 in ppm): 176.1, 170.2, 161.3, 146.5, 135.7, 128.6,128.5, 128.4, 127.8, 69.6, 68.8, 66.9, 51.6, 41.1, 38.6, 31.8, 24.1,19.7, 18.3, 16.0, 11.7.

Example

0.67 g (1.42 mmole) of dipeptide benzyl ester 18 was dissolved in 5 mLdichloromethane. To this solution was added 263 μL of TESCl (236 mg,1.56 mmole, 1.1 eq), and 117 mg (1.72 mmole, 1.2 eq) of imidazole. Thereaction was stirred at 0° C. and solid formed. After 2 hours, the solidwas filtered away and the filtrate was concentrated. The residue was onthe Combiflash (24 g of silica column) with petroleum ether/EtOAC. Afterconcentration, 763 mg (92%) of the desired product 19 was recovered. ¹HNMR (CDCl₃, δ in ppm): 8.12 (s, 1H), 7.46-7.43 (m, 2H), 7.40-7.32 (m,3H), 6.68 (d, J=8.4 Hz, 1H), 5.41 (d, J=12.3 Hz, 1H), 5.36 (d, J=12.3Hz, 1H), 5.13 (t, J=5.7 Hz, 1H), 4.03-3.95 (m, 1H), 3.83 (d, 1H),2.20-2.05 (m, 1H), 1.95-1.86 (m, 2H), 1.48-1.38 (m, 1H), 1.30-1.20 (m,2H), 1.03 (d, 3H), 0.96-0.82 (m, 18H), 0.65 (t, 6H). ¹³C NMR (CDCl₃, δin ppm): 178.2, 168.4, 161.1, 146.5, 135.7, 128.6, 128.5, 128.4, 127.7,70.7, 70.1, 66.9, 51.3, 39.9, 38.3, 31.6, 24.2, 18.3, 17.6, 16.0, 11.5,6.8, 4.6.

Example

746 mg (1.27 mmole) of TES protected dipeptide benzyl ester 19 wasdissolved in 8 mL of THF (anhydrous, inhibitor-free) and cooled to −45°C. After 15 minutes of cooling, 2.8 mL of 0.5 M KHMDS (1.1 eq., 1.4mmole) in toluene solution was added dropwise. After an additional 15mins, 175 μL of chloromethyl butyrate (1.1 eq., 1.4 mmole) was addeddropwise. After 30 mins, TLC showed only a trace amount of startingmaterial left. After 2 hours, the reaction mixture was quenched 1 mLMeOH, and allowed to warm to room temperature. The reaction wasextracted with EtOAc/brine. The organic layer was washed with brine andthen concentrated to give 759 mg (87%) of crude product 20. LCMS:[M+Na]⁺ m/z=710.57. ¹H NMR (CDCl₃, δ in ppm) 8.10 (s, 1H), 7.44-7.40 (m,2H), 7.39-7.30 (m, 3H), 5.43 (d, J=12.3 Hz, 1H), 5.37 (d, J=12.3 Hz,1H), 5.35 (s, 2H), 4.98 (t, J=5.1 Hz, 1H), 4.40-4.20 (br, 1H), 3.52 (d,J=16.0 Hz, 1H), 2.42-2.38 (t, J=6.7 Hz, 2H), 2.25-2.05 (m, 2H),1.78-1.72 (m, 2H), 1.68-1.55 (m, 3H), 1.30-1.20 (m, 1H), 1.00-0.85 (m,24H), 0.65 (t, 6H). ¹³C NMR (CDCl₃, δ in ppm) 177.6, 173.0, 171.0,161.1, 146.6, 135.7, 128.6, 128.42, 128.36, 127.6, 77.2, 70.8, 66.8,63.5, 40.9, 35.9, 34.9, 31.1, 25.0, 20.1, 19.5, 18.1, 15.7, 13.6, 10.5,6.8, 4.7.

Example

239 mg of MEP (1.67 mmole, 1.5 eq), 316 mg of EDC (1.65 mmole, 1.5 eq),and 300 mg of pentafluorophenol (1.63 mmole, 1.5 eq) were dissolved in 8mL of N-methyl-2-pyrrolidone. The reaction was stirred overnight. 759 mg(1.1 mmole) of the alkylated dipeptide 20 in 1 mL NMP was then added. Anadditional 0.8 mL of NMP was used to rinse the flask/syringe to transferresidue to the hydrogenation flask. 60 mg (0.05 eq) of 10% Pd/C was thenadded and the reaction mixture was hydrogenated at 35 PSI, overnight.LC/MS showed the major product is the benzyl ester, along with 10% freeacid. The reaction was filtered through celite, and the filter cake waswashed with EtOAc. The filtrate was extracted with brine, washed withbrine, and concentrated. Combiflash purification with petroleumether/EtOAc resulted in the recovery of 215 mg (25%) of benzyl ester 21.LCMS: [M+H]⁺ m/z=787.66. ¹H NMR (CDCl₃, δ in ppm): 8.09 (s, 1H),7.44-7.40 (m, 2H), 7.39-7.30 (m, 3H), 7.07 (d, J=15.5 Hz, 1H), 5.93 (d,J=12.3 Hz, 1H), 5.42 (d, J=12.3 Hz, 1H), 5.34 (s, 2H), 4.93 (dd, J=8.4,2.7 Hz, 1H), 4.70-4.60 (m, 1H), 4.50-4.30 (br, 1H), 2.88 (m, 1H),2.60-2.28 (m, 4H), 2.21 (s, 3H), 2.08-1.89 (m, 4H), 1.80-1.40 (m, 8H),1.36-1.1.07 (m, 3H), 1.00-0.80 (m, 21H), 0.77 (d, 3H), 0.65 (t, 6H). ¹³CNMR (CDCl₃, 6 in ppm): 177.5, 175.1, 174.1, 173.0, 161.1, 146.5, 135.8,128.6, 128.4, 128.3, 127.6, 77.2, 70.7, 69.5, 69.2, 66.7, 57.3, 55.4,53.5, 53.4, 44.8, 41.3, 36.8, 35.9, 31.4, 30.3, 25.0, 24.7, 23.2, 20.2,19.4, 18.1, 16.2, 13.6, 10.6, 6.8, 5.1, 4.7.

Example Synthesis of EC1759

Paraformaldehyde (1.5 g, 1.25 eq) was added to 16 mL of TMSBr. Theresulted suspension was cooled to 0° C., and 1-pentanol (4.36 mL, 40mmole, 1 equiv.) was added dropwise. The reaction was stirred at 0° C.and warmed up to room temperature. After overnight, TMSBr was evaporatedunder reduced pressure. Vacuum distillation of the residue was carriedout at 7 mm Hg pressure, the fraction came out at 56° C. was the desiredproduct EC1759 (4.3 g, 59%).

Example Synthesis of EC1760

1.58 g (3.09 mmole) TES-dipeptide EC0997 was dissolved in 12 mL THF(anhydrous, inhibitor-free). The resulted solution was cooled to −45° C.To the solution, 6.5 mL of 0.5 M KHMDS in toulene (3.25 mmole, 1.05equiv.) was added dropwise. After finishing the addition, the reactionmixture was stirred at −45° C. for 15 minutes. 600 μL of bromomethylpentyl ether EC1759 (4.1 mmole, 1.33 equiv.) was added dropwise. Thereaction mixture was warmed from −45° C. to −10° C. in 90 minutes, thenquenched with 10% NaCl/1% NaHCO₃ aqueous solution, extracted with EtOAc.The organic phase was washed with 10% NaCl/1% NaHCO₃ aqueous solutionthree times, then brine. The separated organic phase was dried overNa₂SO₄. Na₂SO₄ was filtered off and the solvent was evaporated undervacuum to give 2.4 g of crude product. The crude product was purifiedwith EtOAc/petroleum ether to give 1.47 g of product EC1760 (78%)

Example Synthesis of EC1761

0.38 g of MEP (2.65 mmole, 1.4 equiv.) was suspended in 1.2 mL NMP, 0.53g of PFP (2.88 mmole, 1.5 equiv.) and 0.55 g of EDC (2.87 mmole, 1.5equiv.) were added. The reaction mixture was stirred overnight in ahydrogenation vessel. 1.17 g (1.91 mmole) of alkylated dipeptide EC1760was dissolved in 0.3 mL NMP and transferred to the above hydrogenationvessel, and the residue of the dipeptide was rinsed with 0.3 mL NMP andtransferred to the hydrogenation vessel. 154 mg of 10% Pd/C (dry, 0.05equiv.) was added to the solution. The hydrogenation was carried out at35 PSI. After 5 hrs, LC/MS showed there was no starting material. Thereaction mixture was filtered through celite pad and the reaction vesselwas washed with EtOAc and filtered through celite pad. The combinedsolution was washed with 10% NaCl/1% Na₂CO₃ solution to remove PFP, thenwith brine. The organic phase was dried over Na₂SO₄. Na₂SO₄ was filteredoff and the solvent was evaporated under vacuum to give 1.20 g (88%) ofcrude product EC1761.

Example Synthesis of EC1602

1.17 g (1.65 mmole) of tripeptide ester EC1761 was dissolved in 15 mLMeOH, the solution was cooled to 0° C. 300 mg of LiOH hydrate (7.15mmole, 4.3 equiv.) dissolved in 5 mL H₂O was added to the estersolution, the resulted reaction mixture was stirred and warmed up toroom temperature in 2 hours. LC/MS showed no starting material left.MeOH was removed using rotary evaporator, and the residual was worked upby extraction between EtOAc/brine. The organic phase was dried overNa₂SO₄. Na₂SO₄ was filtered off and the solvent was evaporated undervacuum to give 0.80 g (83%) of crude product EC1602.

Example Synthesis of EC1633

0.80 g (1.37 mmole) of tripeptide acid EC1602 was dissolved in 6.4 mL ofpyridine, the solution was cooled to 0° C. 6.0 mg (0.049 mmole, 0.035equiv) DMAP was added and then 2 mL of acetic anhydride (21.2 mmole,15.5 equiv) was added, the reaction mixture was warmed up to roomtemperature in 5 hours and stored in −20 0° C. for 2 days. 20 mLdioxane/20 mL H2O was added to the reaction mixture at 0° C. and stirredfor 1 hour. The solvent was evaporated under reduced pressure. 20 mL ofphosphate buffer (20 mM) and 5 mL acetonitrile were added to theresidue, the pH of the resulted solution was adjusted to 5.4 usingsaturated NaHCO₃ solution. The solution was loaded on Biotage 120 g C18column. The flask containing the crude product was rinsed with 1 mLacetonitrile/5 mL phosphate buffer and loaded on the column. Thepurification was done using a gradient from 20% ACN/80% water to 70%ACN/30%. The fractions containing the desired product were combined andACN was evaporated under reduced pressure. There were white precipitatecoming out from solution, brine was added to the suspension and EtOAcwas used to extract the desired product. The organic phase was driedover Na₂SO₄. Na₂SO₄ was filtered off and the solvent was evaporatedunder vacuum to give 0.49 g (57%) of product EC1633.

Example General Procedures Synthesis of EC1623 (Scheme 2)

EC1008 (I: R₁=n-propyl. 103 mg) was dissolved in anhydrousdichloromethane (DCM, 2.0 mL) and to this solution was addedtrifluoroacetic acid (TFA, 0.50 mL). The resulting solution was stirredat ambient temperature under argon for 20 minutes, and to which wasadded 1-pentanol (0.72 mL). The reaction mixture was stirred at ambienttemperature for 3 minutes, concentrated on a Buchi Rotavapor at 30° C.for 10 minutes, residue stirred at ambient temperature under high vacuumfor 75 minutes, and to which was added saturated NaHCO₃ solution (10 mL)with vigorous stirring, followed by addition of acetonitrile (ACN, 3.0mL). The resulting white suspension was stirred at ambient temperaturefor 3 minutes and let stand to settle. The top clear solution was loadedonto a Biotage SNAP 12 g KP-C18-HS column on a Biotage system. The whitesolid left in the reaction flask was dissolved in water (5.0 mL) and thesolution was also loaded onto the Biotage column. The remaining solidstuck on the glass wall of the reaction flask was dissolved in ACN (2.0mL). To this solution was added water (6.0 mL) and the resulting cloudysolution was loaded onto the same Biotage column. The reaction mixturewas eluted following these parameters: Flow rate: 15 mL/min. A: water;B: CAN. Method: 25% B 2 CV (column volume), 25-50% B 3 CV, and 50% B 5CV (1 CV=15 mL). Fractions containing the desired product was collectedand freeze-dried to afford EC1623 (II: R=n-pentyl. 95.9 mg) as a whitepowder.

Example Synthesis of EC1662 (Scheme 3)

Step 1: Anhydrous DCM (5.0 mL) was added to a mixture of EC1623 (II:R=n-pentyl. 114 mg), pentafluorophenol (PFP, 67.3 mg), and DCC-resin(2.3 mmol/g, 396 mg) and the suspension was stirred at ambienttemperature under argon for 23 hours. The resin was filtered off andwashed with anhydrous DCM (3.0 mL) and the combined filtrates wereconcentrated under reduced pressure to give a residue, which wasvacuumed at ambient temperature for 1 hour prior to use in Step 3.

Step 2: EC1426 (114 mg) was dissolved in anhydrous DCM (1.5 mL) and towhich was added TFA (0.50 mL). The resulting solution was stirred atambient temperature under argon for 70 minutes and concentrated underreduced pressure to give a residue, which was co-evaporated withanhydrous DCM (2.0 mL×3) and vacuumed at ambient temperature for 9 hoursprior to use in Step 3.

Step 3: The residue from Step 1 was dissolved in anhydrous DCM (1.5 mL)and to this solution was added DIPEA (0.50 mL) followed by a solution ofthe residue from Step 2 dissolved in anhydrous dimethylformamide (DMF,1.5 mL). The resulting solution was stirred at ambient temperature underargon for 1 hour, diluted with ethyl acetate (EtOAc, 60 mL), and washedwith brine (20 mL×3). The organic layer was separated, dried (Na₂SO₄),and concentrated under reduced pressure to give a residue, which wasvacuumed at ambient temperature for 2 hours, dissolved in DCM (3.5 mL),and loaded onto a 24 g silica gel column on a CombiFlash system forpurification. The materials were eluted with 0-5% MeOH in DCM to affordEC1662 (III: R=n-pentyl. 171 mg) as a white solid.

Synthesis of EC1664 (Scheme 3)

A solution of EC1454 (SPACER-SH; See FIG. 1 for structure. 44.1 mg.) in20 mM phosphate buffer (pH 7.0, 4.0 mL) was added to a solution ofEC1662 (24.1 mg) in MeOH (4.8 mL), followed by addition of saturatedNa₂SO₄ (0.30 mL). The reaction mixture was stirred at ambienttemperature under argon for 30 minutes and the solution was injectedonto a preparative HPLC (A: 50 M NH₄HCO₃ buffer, pH 7.0; B: CAN. Method:10-80% B in 20 minutes.) for purification. Fractions containing thedesired product were collected and freeze-dried to afford EC1664 (IV:R=n-pentyl. 42.8 mg) as a fluffy yellow solid.

1. A process for preparing a compound of the formula

or a pharmaceutically acceptable salt thereof; wherein Ar₁ is optionallysubstituted aryl or optionally substituted heteroaryl; R₁ is hydrogen,alkyl, arylalkyl or a pro-drug forming group; R₂ is selected from thegroup consisting of optionally substituted alkyl and optionallysubstituted cycloalkyl; R₁₂ is alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, eachof which is optionally substituted; R₃ is optionally substituted alkyl;R₄ is optionally substituted alkyl or optionally substituted cycloalkyl;R₅ and R₆ are each independently selected from the group consisting ofoptionally substituted alkyl and optionally substituted cycloalkyl; R₇is optionally substituted alkyl; and n is 1, 2, 3, or 4; wherein theprocess comprises the step of treating a compound of formula A withtriethylsilyl chloride and imidazole in an aprotic solvent, where R₈ isC1-C6 unbranched alkyl

or the step of treating a compound of formula B with a base and acompound of the formula ClCH₂OC(O)R₂ in an aprotic solvent at atemperature from about −78° C. to about 0° C.; wherein the molar ratioof the compound of the formula ClCH₂OC(O)R₂ to the compound of formula Bfrom about 1 to about 1.5, where R₈ is C1-C6 unbranched alkyl

or the steps of a) preparing a compound of formula (E1), where X₁ is aleaving group, from a compound of formula E

and b) treating a compound of formula C under reducing conditions in thepresence of the compound of formula E1, where R₈ is C1-C6 unbranchedalkyl

or the step of contacting compound D with an alcohol, R₁₂OH, where R₁₂is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl,aryl, arylalkyl or heteroarylalkyl, each of which is optionallysubstituted; and a transesterification catalyst selected from the groupconsisting of (R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂,(R₁₃)₂SnS, (R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃ is independentlyselected from alkyl, arylalkyl, aryl, or cycloalkyl, each of which isoptionally substituted;

or the step of treating the compound AF with a metal hydroxide or ametal carbonate;

or the step of treating a compound of formula BG with an acylating agentof formula R₄C(O)X₂, where X₂ is a leaving group

or the steps of c) forming an active ester intermediate from a compoundof formula AH

and d) reacting the active ester intermediate with a compound of theformula I;

or one or more combinations thereof.
 2. The process of claim 1 whereinR₄ is optionally substituted alkyl.
 3. The process of claim 1 er-2comprising the step of treating a compound of formula A withtriethylsilyl chloride and imidazole in an aprotic solvent, where R₈ isC1-C6 unbranched alkyl


4. The process of claim 1 comprising the step of treating a compound offormula B with a base and a compound of the formula ClCH₂OC(O)R₂ in anaprotic solvent at a temperature from about −78° C. to about 0° C.;wherein the molar ratio of the compound of the formula ClCH₂OC(O)R₂ tothe compound of formula B from about 1 to about 1.5, where R₈ is C1-C6unbranched alkyl


5. The process of claim 1 comprising the steps of a) preparing acompound of formula (E1), where X₁ is a leaving group, from a compoundof formula E

and b) treating a compound of formula C under reducing conditions in thepresence of the compound of formula E1, where R₈ is C1-C6 unbranchedalkyl


6. The process of claim 1 comprising the step of treating compound Dwith an alcohol, R₁₂OH, where R₁₂ is alkyl, alkenyl, alkynyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl orheteroarylalkyl, each of which is optionally substituted; and atransesterification catalyst selected from the group consisting of(R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂, (R₁₃)₂SnS,(R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃ is independently selectedfrom alkyl, arylalkyl, aryl, or cycloalkyl, each of which is optionallysubstituted;


7. The process of claim 1 comprising the step of treating the compoundAF with a metal hydroxide or a metal carbonate;


8. The process of claim 1 comprising the step of treating a compound offormula BG with an acylating agent of formula R₄C(O)X₂, where X₂ is aleaving group


9. The process of claim 1 comprising the steps of c) forming an activeester intermediate from a compound of formula AH

and d) reacting the active ester intermediate with a compound of theformula I


10. The process of claim 1 wherein R₁ is hydrogen, benzyl, or C1-C4alkyl.
 11. The process of claim 1 wherein R₂ is C1-C8 alkyl or C3-C8cycloalkyl.
 12. The process of claim 1 wherein R₂ is CH₂CH(CH₃)₂,CH₂CH₂CH₃, CH₂CH₃, CH═C(CH₃)₂, or CH₃.
 13. The process of claim 1wherein R₃ is C1-C4 alkyl.
 14. The process of claim 1 wherein Ar₁ isphenyl or hydroxyphenyl.
 15. The process of claim 1 wherein R₄ is C1-C8alkyl or C3-C8 cycloalkyl.
 16. The process of claim 1 wherein R₅ isbranched C3-C6 or C3-C8 cycloalkyl.
 17. The process of claim 1 whereinR₆ is branched C3-C6 or C3-C8 cycloalkyl.
 18. The process of claim 1wherein R₇ is C1-C6 alkyl.
 19. The process of claim 1 wherein R₁₂ isCH₂CH═CH₂, or CH₂(CH₂)nCH₃, where n=1, 2, 3, 4, 5, or
 6. 20. The processof claim 1 wherein the metal hydroxide is LiOH.